Optimized minizymes and miniribozymes and uses thereof

ABSTRACT

This invention is directed to improved catalytic compounds, minizymes and miniribozymes, capable of hybridizing with a target RNA to be cleaved. The minizymes and miniribozymes and compositions of the present invention may be used in vitro or in vivo. They may be used as diagnostic or therapeutic agents.

This application is a continuation-in-part of U.S. Ser. No. 08/488,181, filed Jun. 7, 1995 the contents of which are hereby incorporated by reference into the subject application.

Throughout this application various references are cited in bracket by author and publication year. The full citations are listed alphabetically and may be found immediately preceding the claims. These publications are hereby incorporated by reference into the present application.

BACKGROUND OF THE INVENTION

Several types of ribozymes have been identified in living organisms. One of the first ribozymes to show catalytic turnover was the RNA moiety of ribonuclease P. Ribonuclease P (RNase P) cleaves precursor tRNAs (pre-tRNAs) at their 5' ends to give the mature 5'-termini of tRNAs. In Escherichia coli and Bacillus subtilis, the RNase P holoenzyme is composed of one basic protein subunit of approximate M_(r) 14,000 (119 amino acids) and one single stranded RNA molecule of 377 and 401 nucleotides, respectively [Baer, 1990; Altman 1987; Waugh, 1989; Pace, 1990; Nichols, 1988]. Another early ribozyme to show cleavage was the L-19 intervening sequence (IVS) from tetrahymena. The 413 nucleotide intervening sequence (IVS) in the nuclear rRNA precursor from Tetrahymena thermophila can be excised and the two exons ligated in the complete absence of any protein [Kruger, 1982; Cech, 1981]. Unique to this class of self-splicing reaction is the requirement of a guanosine or 5' guanosine nucleotide cofactor. The hammerhead, which in nature undergoes a self-cleavage reaction, constitutes a third class of ribozymes. A number of plant pathogenic RNAs [Symons, 1989; Symons, 1990; Bruening, 1989; Bruening 1990], one animal viral RNA [Taylor, 1990] and a transcript from satellite II of DNA of the newt [Epstein, 1987; Epstein 1989] and from a Neurospora DNA plasmid [Saville, 1990] undergo a site specific self-cleavage reaction in vitro to produce cleavage fragments with a 2',3'-cyclic phosphate and a 5'-hydroxyl group. This reaction is unlike RNase P RNA cleavage of pre-tRNAs, where the internucleotide bond undergoes a phosphoryl transfer reaction in the presence of Mg⁺⁺ or other divalent cations. Metal cations may be essential to RNA catalysis [Pyle, 1993]. Other reactions documented to date show that ribozymes can catalyze the cleavage of DNA [Robertson, 1990; Herschlag 1990], the replication of RNA strands [Green, 1992], the opening of 2'-3'-cyclic phosphate rings [Pan, 1992], as well as react with phosphate monoesters [Zaug, 1986] and carbon centers [Noller, 1992; Piccirilli, 1992]. Finally, ribozymes with new kinds of catalytic reactivity are being created through techniques of in vitro selection and evolution [Breaker and Joyce, 1994; Szostak, 1992].

The ability to design a ribozyme to specifically target and cleave any designated RNA sequence has led to much interest in the potential application of hammerhead ribozymes in transgenic plants and in animal health as gene therapy agents or drugs. To improve the ability to treat a disease or target a specific nucleic acid, it is desirable to optimize the ribozyme to achieve the maximum cleavage activity. While much success has been achieved in vitro in targeting and cleaving a number of designated RNA sequences (Saxena and Ackerman, 1990; Lamb and Hay, 1990; Evans, et al., 1992; Mazzolini, et al., 1992; Homann, et al., 1993), there are fewer whole cell examples.

Previous reports have demonstrated that high levels of ribozyme expression are required to achieve reduced accumulation of target sequence in vivo [Cameron and Jennings, 1989; Cotten and Birnsteil, 1989; Sioud and Drilca, 1991; L'Huillier, et al., 1992; Perriman et al., 1993]. Another article suggests a necessity for the target and ribozyme to be sequestered in the same cellular compartment [Sullenger and Cech, 1993]. These reports demonstrate that hammerhead ribozymes are clearly capable of specific cleavage of a designated target RNA within a biological system.

SUMMARY OF THE INVENTION

This invention is directed to improved catalytic compounds, minizymes and miniribozymes, capable of hybridizing with a target RNA to be cleaved. The minizymes and miniribozymes and compositions of the present invention may be used in vitro or in vivo. They may be used as diagnostic or therapeutic agents.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the CAT Expression (Mean±SEM (Standard Error of the Mean)) for Different treatments. Each experiment is performed in triplicate.

FIG. 2 shows the extent of Alamar Blue Reduction (mean±SEM) in the same experiments as in FIG. 1. Extent of reduction relates to metabolic activity of the cells during the 18 hours post-transfection.

FIG. 3 shows the cleavage of the IL-2 substrate at 37° C. by minizymes (with cctt and gtttt linkers) and a ribozyme with DNA arms.

FIG. 4 shows the numbering scheme of (Hertel 1992) for a hammerhead ribozyme and its substrate (Seq ID No. 1-2).

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to a compound having the formula (SEQ ID No. 2): ##STR1## wherein each X represents a nucleotide which may be the same or different and Q represents guanylate or deoxyguanylate and both of which may be substituted or modified in its sugar, base or phosphate; wherein each of (X)_(n-1) --A and (X)_(n') represents an oligonucleotide having a predetermined sequence which is capable of hybridizing with an RNA target sequence to be cleaved; wherein each of n and n' represents an integer which defines the number of nucleotides in the oligonucleotide such that (X)_(n-1) --A and (X)_(n') are sufficient to hybridize to the target sequence; wherein each solid line represents a chemical linkage providing covalent bonds between the nucleotides located on either side thereof; wherein "a" represents an integer which defines a number of nucleotides with the proviso that "a" may be 0 or 1 and if 0, the A located 5' of (X)_(a) is bonded to the Q located 3' of (X)_(a) ; and wherein (T/U)_(b) represents an oligonucleotide with the proviso that "b" represents an integer which is 3 or 4. Alternatively, the compound may have the formula (Seq ID No. 4): ##STR2## wherein P represents cytidylate or deoxycytidylate. In the compounds above, the oligonucleotide 3'-(X)_(n-1) --A may be 3'-(X)_(n) or 3'-(X)_(n-2) --C--A--. Preferably, (X_(a)) is absent. The integer "b" of (T/U)_(b) is preferably equal to 3 or 4. Preferably, (T/U)_(b) is a (T)_(b).

The invention is also directed to compositions comprising the compounds above in association with an acceptable carrier; the carrier is preferably a pharmaceutically acceptable carrier.

The invention is also directed to an oligonucleotide transfer vector containing a nucleotide sequence which on transcription gives rise to the compound above. The transfer vector may be a bacterial plasmid, a bacteriophage DNA, a cosmid, an eukaryotic viral DNA, a plant DNA virus, a composite geminivirus, a binary plant expression vector (Ri or Ti) an infective phage particle or a portion thereof. The packaged oligonucleotide transfer vector may contain promoter sequences for RNA polymerase II, human tRNA^(val), plant tRNA, human tRNA, snRNA promoter or RNA polymerase III. The invention also includes a host cell transformed by the transfer vector. The host cell is a prokaryotic host cell, an eukaryotic host cell, an E. coli host cell, a monkey COS host cell, a Chinese hamster ovary host cell, a mammalian host cell, a plant host cell, a plant protoplast host cell, a hematopoietic host cell, a stem cell, a hematopoietic progenitor cell, a lymphoid cell, T-cell, a B-cell, pre-B cell, a CD4+T-cell or a peripheral blood mononuclear cell.

The invention also provides a method of cleaving a target mRNA in a subject which comprises administering to the subject an effective amount of the compound above or a vector capable of expressing the compound. The administration may be topical in an amount between 1 ng and 10 mg. The administration may also be systemic and administered in an amount between 1 ng and 500 μg/kg weight/day. The administration may also be aerosol administration. The invention also provides a method of cleaving a target mRNA in a host cell which comprises administering to the host cell an effective amount of the compound above.

The compound above may further comprise an antisense nucleic acid which is capable of hybridizing with an RNA target sequence. The compound above may further comprise at least one additional non-naturally occurring oligonucleotide compound which comprises nucleotides whose sequence defines a conserved catalytic region and nucleotides whose sequence is capable of hybridizing with a predetermined target sequence. The additional non-naturally occurring oligonucleotide compound may be a hammerhead ribozyme, a minizyme, a hairpin ribozyme, a hepatitis delta ribozyme, an RNAase P ribozyme or a combination thereof. See for example hammerhead ribozyme Haseloff et al. U.S. Pat. No. 5,254,678, issued Oct. 18, 1993; Jennings U.S. Pat. No. 5,298,612, issued Mar. 29, 1994; Group I introns, Cech et al. U.S. Pat. No. 4,740,463, issued Apr. 26, 1988; Altman et al. U.S. Pat. No. 5,168,053, issued Dec. 1, 1992 or PCT International Publication No WO 92/03566), hepatitis delta ribozymes (e.g. Blumenfeld et al. PCT International Application No. WO/90/05157) and hairpin ribozymes (European Patent Application No. EP 360,257, Hampel et al. Nuc. Acids Res. (1990) 18:299-304).

Preferred cleavage sites in the target RNA have the sequence "XUX", preferably GUC, GUU, GUA, UUA and TUC. By way of example, suitable reaction conditions may comprise a temperature from about 4 degree(s) C. to about 60 degree(s) C., preferably from about 10 degree(s) to 45 degree(s) C., more preferably from about 20 degree(s) to 43 degree(s) C., pH from about 6.0 to about 9.0 and concentration of divalent cation (such as Mg²⁺) from about 1 to about 100 mM (preferably 1 to 20 mM). The nucleotides of the sequences (X)_(n) and (X)_(n') of the compounds above may be of any number and sequence sufficient to enable hybridization with the nucleotides in the target RNA, as described herein. Ribozymes containing a small number of nucleotides in each of the groups (X)_(n) and (X)_(n') of the compounds above (such as four nucleotides) would generally be incubated at lower temperatures, such as about 20 degree(s) C. to about 25 degree(s) C. to aid hybridizing with the nucleotide sequences in the substrate. The number of nucleotides n and n' in (X)_(n) and (X)_(n') are not necessarily equal. The invention is also directed to covalently-linked multiple ribozymes, where each ribozyme is directed to a target sequence which may be the same or different. In addition these compounds may be covalently attached to an antisense molecule which may be 10 to 100 bases in length. Antisense sequences capable of hybridizing to an RNA in a mammal or plant are well known see (Shewmaker et al. U.S. Pat. No. 5,107,065, issued Apr. 21, 1992). As the ribozyme acts as an enzyme, showing turnover, the ratio of ribozyme to substrate may vary widely.

A target RNA containing a suitable cleavage site such as XUX site may be incubated with a compound described above. The nucleotide sequences (X)_(n) and (X)_(n') of the compounds above are selected to hybridize with their substrate. They may be selected so as to be complementary to nucleotide sequences flanking the cleavage site in the target RNA. On incubation of the ribozyme or ribozyme composition and its substrate, an enzyme/substrate complex is formed as a result of base pairing between corresponding nucleotides in the ribozyme and the substrate. Nucleotide sequences complementary to (X)_(n) and (X)_(n') of the compounds above flanking the cleavage site in the substrate may form a double stranded duplex with (X)_(n) and (X)_(n') as a result of base pairing, which base pairing is well known in the art [See for example: Sambrook, 1989]. The formation of a double stranded duplex between the nucleotides may be referred to as hybridization [Sambrook, 1989]. The extent of hybridization or duplex formation between the ribozyme and its substrate can be readily assessed, for example, by labeling one or both components, such as with a radiolabel, and then subjecting the reaction mixture to polyacrylamide gel electrophoresis under non-denaturing conditions [Sambrook, 1989]. If the target is cleaved specifically on incubation with the compound, the compound is active and falls within the scope of this invention. Accordingly, a ribozyme containing substituted or modified nucleotides in the conserved region may be simply tested for endonuclease activity in a routine manner.

As will be readily appreciated by workers in the field to which this invention relates, the cleavage of a target RNA may be readily assessed by various methods well known in the art [See for example: Sambrook, 1989]. Cleavage may, for example, be assessed by running the reaction products (where the substrate is radioactively labeled) on acrylamide, agarose, or other gel systems under denaturing conditions, and then subjecting the gel to autoradiography or other analytical technique to detect cleavage fragments [Sambrook, 1989].

In another embodiment, the invention provides a composition which comprises the compounds above in association with an acceptable carrier.

The invention is also directed to an oligonucleotide transfer vector containing a nucleotide sequence or sequences which on transcription gives rise to the compounds above. The transfer vector may be a bacterial plasmid, a recombinant bacterial plasmid, a bacteriophage DNA, a cosmid, or an eukaryotic viral DNA. The transfer vector may also contain an appropriate transcription promoter sequence such as that for RNA polymerase II, RNA polymerase III, a viral promoter such as SV40 or HIV LTR, a plant promoter such as CaMV S35 or a promoter associated with animal health. The vector may also contain an appropriate termination sequence. Preferably, the plant or animal promoter is capable of expression in a regulated manner. Such promoter control regions would be regulated by endogenous signals to direct either tissue specific or temporal expression or by externally administered compounds to elicit transcription of downstream sequences. It may also contain sequences to effect integration into the host genome on episomal replication in the host cell.

The invention also provides a host cell transformed by the transfer vector as mentioned above, which may be a prokaryotic host cell or an eukaryotic host cell e.g. yeast cell or yeast protoplast, E. coli host cell, a monkey host cell (e.g. COS), a Chinese hamster ovary host cell, a mammalian host cell, a plant host cell, or a plant protoplast host cell.

In one embodiment, there is provided a packaged oligonucleotide transfer vector, as mentioned hereinabove, which is a plant virus, a composite mammalian virus, a geminivirus, a Ti or Ri plasmid, an infective phage particle or portion thereof.

In another embodiment, the composition, as discussed above, is in association with an acceptable carrier. This invention also provides a composition as discussed hereinabove wherein the oligonucleotide is an oligoribonucleotide or an RNA-DNA hybrid molecule comprising nucleotides which may be substituted or modified in their sugar, base or phosphate group. It is preferred that the oligonucleotide be an oligoribonucleotide or a hybrid RNA-DNA molecule. However, other substitutions or modifications in the nucleotide are possible providing that endonuclease activity is not lost. Such derivatives or modifications are described below.

The nucleotides may be in the form of deoxyribonucleotides, ribonucleotides, deoxyribonucleotide ribonucleotide hybrids, or derivatives thereof as herein described. The flanking sequences (X)_(n) and (X)_(n') may be chosen to optimize stability of the ribozyme from degradation. For example, deoxyribonucleotides are resistant to the action of ribonucleases. Modified bases, sugars or phosphate linkages of nucleotides, such as phosphoramidate, or phosphorothioate linkages in the sugar phosphate chain of X_(n') and X_(n'), may also provide resistance to nuclease attack. Binding affinity may also be optimized in particular circumstances, by providing nucleotides solely in the form of ribonucleotides, deoxyribonucleotides, or combinations thereof. In some circumstances it may be necessary to optimize the composition of the sequences (X)_(n) and (X)_(n'), to maximize target RNA cleavage. The cleavage activity of ribozymes having flanking nucleotide sequences which hybridize to target sequences and which are comprised wholly of deoxyribonucleotides may, in some circumstances, have reduced activity. In such circumstances optimization may involve providing a mixture of deoxyribonucleotides and ribonucleotides in the nucleotide sequences (X)_(n) and (X)_(n'). For example, nucleotides in the ribozyme which are proximal to the cleavage site in a target RNA may be in the form of ribonucleotides.

The respective 3' and 5' termini of the sequences (X)_(n) and (X)_(n') or alternatively the 3' and 5' end termini of the ribozyme, may be modified to stabilize the ribozyme from degradation. For example, blocking groups may be added to prevent terminal nuclease attack, in particular 3'-5' progressive exonuclease activity. By way of example, blocking groups may be selected from substituted or unsubstituted alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted alkanoyl. Substituents may be selected from C₁ -C₅ alkyl; halogens such as F, Cl or Br; hydroxy; amino; C₁ -C₅ alkoxy and the like. Alternatively, nucleotide analogues such as phosphorothioates, methylphosphonates or phosphoramidates or nucleoside derivatives (such as alpha--anomer of the ribose moiety) which are resistant to nuclease attack may be employed as terminal blocking groups. The blocking group may be an inverted linkage such as a 3' 3' thymidine linkage or a 5' 5' pyrophosphate linkage as in the guanosine cap.

Alternatively, groups which alter the susceptibility of the ribozyme molecule to other nucleases may be inserted into the 3' and/or 5' end of the ribozyme. For example, 9-amino-acridine attached to the ribozyme may act as a terminal blocking group to generate resistance to nuclease attack on the ribozyme molecules and/or as an intercalating agent to aid endonucleolytic activity. It will be readily appreciated that a variety of other chemical groups, e.g. spermine or spermidine could be used in a related manner.

It is also possible to stabilize the ribozyme from degradation by embedding it in an RNA molecule. These molecules can be produced either in vitro or in vivo by DNA coding sequences being operably linked to transcriptional control sequences as appropriate. Examples of RNA molecules into which ribozymes could be inserted may include, but are not limited to, tRNA, mRNA, rRNA, snRNA or other RNA molecules. In addition, the ribozyme may be inserted into an engineered stable stem loop structure. The compound may also be coupled with other stabilizing structures such as a transcription terminator on the 3' end such as the T7 terminator, ρ-independent terminator, cry element (Gelfand et al. U.S. Pat. No. 4,666,848, issued May 19, 1987) or the TrpE terminator. Furthermore, sequences such as the poly(A) addition signal AATAAA may be added. In addition, strategies involving changing the length of the 3' non-coding region may be used (see Gillies, U.S. Pat. No. 5,149,635, issued Sep. 22, 1992). Alternatively, a stabilizing sequence or protein binding domain such as Sioud, PCT International application WO 94/10301 may be used. Further, it is possible to insert the compound into a DNA molecule as well.

The compounds of this invention may be covalently or non-covalently associated with affinity agents such as proteins, steroids, hormones, lipids, nucleic acid sequences, intercalating molecules (such as acridine derivatives, for example 9-amino acridine) or the like to modify binding affinity for a substrate nucleotide sequence or increase affinity for target cells, or localization in cellular compartments or the like. For example, the ribozymes of the present invention may be associated with RNA binding peptides or proteins which may assist in bringing the ribozyme into juxtaposition with a target nucleic acid such that hybridization and cleavage of the target sequence may take place. Nucleotide sequences may be added to the respective 3' and 5' termini of the sequences (X)_(n) and (X)_(n') or alternatively the 3' and 5' end termini of the ribozyme to increase affinity for substrates. Such additional nucleotide sequences may form triple helices with target sequences [Strobel, 1991] which may enable interaction with an intramolecularly folded substrate. Alternatively, modified bases (non-natural or modified bases as described in Principles of Nucleic Acid Structure [Saenger, 1984]) within the additional nucleotide sequences may be used that will associate with either single stranded or duplex DNA generating base pair, triplet, or quadruplet, interactions with nucleotides in the substrate. Suitable bases would include inosine, 5-methylcytosine, 5-bromouracil and other such bases as are well known in the art, as described, for example, in Principles of Nucleic Acid Structure [Saenger, 1984].

The compounds of this invention may be produced by nucleotide synthetic techniques which are known in the art, and described for example by Carruthers et al., Foehler et al. and Sproat et al. [Carruthers, 1987; Foehler, 1986; Sproat, 1984]. Generally, such synthetic procedures involve the sequential coupling of activated and protected nucleotide bases to give a protected nucleotide chain, whereafter protecting groups may be removed by suitable treatment. Preferably the compounds will be synthesized on an automated synthesizer such as those made by Applied Biosystems (a Division of Perkin Elmer), Pharmacia or Millipore. Alternatively, the ribozymes in accordance with this invention may be produced by transcription of nucleotide sequences encoding said ribozymes in host-cells or in cell free systems utilizing enzymes such as T3, SP6 or T7 RNA-polymerase.

In addition to being synthesized chemically, ribozymes with modified nucleotides may be synthesized enzymatically. The phosphodiester bonds of RNA can be replaced by phosphorothioate linkages by in vitro transcription using nucleoside α-phosphorthiotriphosphates. T7 RNA polymerase specifically incorporates the Sp isomer of α-phosphorthiotriphosphate with inversion of configuration to produce the Rp isomer of the phosphorothioate linkage. The methods to produce transcripts fully substituted with phosphorothioate linkages adjacent to a given nucleotide, or to produce partially substituted transcripts containing approximately one phosphorothioate linkage per molecule, are described by Ruffner and Uhlenbeck (1990). Conrad et al. (1995) describe methods of using T7 RNA polymerase to produce chimeric transcripts containing ribonucleotides and deoxyribonucleotides (with and without phosphorothioate linkages), and also ribonucleotides and 2'-O-methylnucleotides (with and without phosphorothioate linkages). These methods have been shown to produce transcripts containing up to 50% deoxyribonucleotides, and up to 58% 2'-O-methylnucleotides. Aurup et al (1992) describe methods for using T7 polymerase to produce transcripts containing 2'-fluoro-2'-deoxyuridine, 2'-fluoro-2'-deoxycytidine, and 2'-amino-2'deoxyuridine. (Aurup, 1992; Conrad, 1995; Ruffner, 1990) Further means for producing the ribozymes of this invention are further discussed below [Sambrook, 1989].

Nucleotides represented in the compounds above comprise a sugar, base, and a monophosphate group or a phosphodiester linkage. Accordingly, nucleotide derivatives or modifications may be made at the level of the sugar, base, monophosphate groupings or phosphodiester linkages. It is preferred that the nucleotides in the compounds above be ribonucleotides or RNA/DNA hybrids, however, other substitutions or modifications in the nucleotide are possible providing that endonuclease activity is not lost.

In one aspect of this invention, the sugar of the nucleotide may be a ribose or a deoxyribose such that the nucleotide is either a ribonucleotide or a deoxyribonucleotide, respectively. Furthermore, the sugar moiety of the nucleotide may be modified according to well known methods in the art [See for example: Saenger, 1984; Sober, 1970]. This invention embraces various modifications to the sugar moiety of nucleotides as long as such modifications do not abolish cleavage activity of the ribozyme. Examples of modified sugars include replacement of secondary hydroxyl groups with halogen, amino or azido groups; 2'-alkylation; conformational variants such as the O2'-hydroxyl being cis-oriented to the glycosyl C₁ '-N link to provide arabinonucleosides, and conformational isomers at carbon C₁ ' to give alpha -nucleosides, and the like. In addition, the invention is directed to compounds with a substituted 2' hydroxyl such as 2' O-allyl, or 2' O-methyl. Alternatively, the carbon backbone of the sugar may be substituted such as in 2' C-allyl.

Accordingly, the base of the nucleotide may be adenine, 2-amino adenine, cytosine, guanine, hypoxanthine, inosine, methyl cytosine, thymine, xanthine, uracil, or other methylated bases.

Nucleotide bases, deoxynucleotide bases, and ribonucleotide bases are well known in the art and are described, for example in Principles of Nucleic Acid Structure [Saenger, 1984]. Furthermore, nucleotide, ribonucleotide, and deoxyribonucleotide derivatives, substitutions and/or modifications are well known in the art [See for example: Saenger, 1984; Sober, 1970], and these may be incorporated in the ribozyme made with the proviso that endonuclease activity of the ribozyme is not lost. As mentioned previously, endoribonuclease activity may be readily and routinely assessed.

In addition, a large number of modified bases are found in nature, and a wide range of modified bases have been synthetically produced [See for example: Saenger, 1984; Sober, 1970]. For example, amino groups and ring nitrogens may be alkylated, such as alkylation of ring nitrogen atoms or carbon atoms such as N₁ and N₇ of guanine and C₅ of cytosine; substitution of keto by thioketo groups; saturation of carbon-carbon double bonds, and introduction of a C-glycosyl link in pseudouridine. Examples of thioketo derivatives are 6-mercaptopurine and 6-mercaptoguanine. Bases may be substituted with various groups, such as halogen, hydroxy, amine, alkyl, azido, nitro, phenyl and the like. The phosphate moiety of nucleotides or the phosphodiester linkages of oligonucleotides are also subject to derivatization or modifications, which are well known in the art. For example, replacement of oxygen with nitrogen, sulphur or carbon gives phosphoramidates, (phosphorothioates, phosphorodithioates) and phosphonates, respectively. Substitutions of oxygen with nitrogen, sulphur or carbon derivatives may be made in bridging or non bridging positions. It has been well established from work involving antisense oligonucleotides [Uhlman, 1990] that phosphodiester and phosphorothioate derivatives may efficiently enter cells (particularly when of short length), possibly due to association with a cellular receptor. Methylphosphonates are probably readily taken up by cells by virtue of their electrical neutrality.

A further aspect of the invention provides alternative linkages such as an amide, a sulfonamide, a hydroxylamine, a formacetal, a 3'-thioformacetal, a sulfide, or an ethylene glycol function to replace the conventional phosphodiester linkage. These modifications may increase resistance towards cellular nucleases and/or improved pharmacokinetics.

Information on Synthesis of Protected Nucleotides and their Incorporation into Modified Ribozymes.

POSSIBLE NUCLEOTIDE MODIFICATIONS

Sugar Modifications may be 2' fluoro, 2' amino, 2' O-allyl, 2' C-allyl, 2' O-methyl, 2' O-alkyl, 4'-thio-ribose, α-anomer, arabinose, other sugars, or non-circular analogues.

Phosphate Modifications may be phosphorothioate (non-bridging), phosphorodithioate (non bridging), 3' bridging phosphorothioate, 5' briding phosphorothioate, phosphoramidates, 3' bridging phosphoramidate, 5' bridging phosphoramidate, methyl phosphonate, other alky phosphonates or phosphate triesters.

The phosphodiester linkage may be replaced by an amide, carbamate, thiocarbamate, urea, amine, hydroxylamine, formacetal, thioformacetal, allyl ether, allyl, ether, thioether, or PNA (peptide nucleic acid) linkage.

Modifications in base may be purine, 2,6-diaminopurine, 2-aminopurine, O⁶ -methylguanosine, 5-alkenylpyrimidines, 5-propyne, inosine, 5-methylcytosine, pseudouridine, abasic (ribose or deoxyribose).

Some nucleotides may be replaced with the following chemical linkers: 1,3-propane-diol, alkane-diols, or various polymers of (ethyleneglycol, tetraethylene glycol, hexaethyleneglycol).

Other Modifications to the 3' end may be selected from: 3'--3' inverted linkage (inverted diester or inverted phosphoramidate). 3'--3' linked abasic ribose, or end-capped (methoxyethylamine phosphoramidate).

Modified sugars may be synthesized as follows: 2'-deoxy-2'-fluoro uridine (Sinha, 1984); 2'-deoxy-2' fluoro cytidine (Sinha, 1984); 2'-deoxy-2' fluoroadenosine; synthesis and incorporation into ribozyme (Olsen, 1991); 2'-deoxy-2'-amino uridine and 2'-deoxy-2'-amino cytidine (Heidenreich, 1994); 2'-O-allyl-(uridine or cytidine or adenosine or guanosine) (Available from Boehringer Mannheim, Mannheim, Germany) or (Badger, 1994). 2'-deoxy-2'-C-allyl-ribonucleotides; 2'-O-methyl ribonucleotides see Review: (Sproat, B. S., 1991A) (also Available from Chemgenes, Waltham, Mass. or Glen Research, Sterling, Va. other 2'-O-alkyl-ribonucleotides, Synthesis see (Monia, B. P., 1993; Sproat, B. S., 1991B); α-anomer of uridine, cytidine, adenosine and guanosine, see (Debart, F., 1992 and references therein); Other modified sugars, etc. Arabinose (Garbesi, A., 1993); Hexose-thymidine (Augustyns, K., 1992) and linear analogues of sugars (Hendry, 1994).

Modified phosphates may be synthesized as follows: Phosphorothioate; synthesized by modification of oxidation procedure during phosphoramidite synthesis. Reagents commercially available from Perkin Elmer and others, products are mixture of isomers, some methods available for stereospecific synthesis of phosphorothioate, see ref: (Stec, 1991); Phosphorodithioate; (Eldrup, A. B., 1994; Caruthers, 1991; Beaton, 1991); 3'-bridging phosphorothioate; 5' bridging phosphorothioate; phosphoramidates (non-bridging, oxidize the phosphite triester with solution containing the required amine); (Froehler, B., 1988; Jager, A., 1988; Letsinger, R. L., 1988); 3' bridging phosphoramidate (NH replaces 3' O) (Forms very stable duplexes) (Letsinger, 1992; Gryaznov, S. M., 1995; Chen, J. K., 1995); 5' Bridging Phosphoramidate (NH replaces 5' O; thymidine analogue only, weak binder) (Gryaznov, S. M., 1992); Methylphosphonate (reagents are commercially available; Glen Research or Chemgenes Stereospecific; Rp isomers bind stronger: (Savchenko, 1994; Miller, 1991); 5'-deoxy, 5'-methylphosphonate (Szabo, 1995); Other alkyl-phosphonates (Fathi, 1994A; Fathi, 1994B); Phosphate triesters(Summers, 1986).

Replacements for the Phosphodiester Linkage may be synthesized as follows:

For review see (De Mesmaker, 1995) Amides (Chur,1993; Blommers, 1994; De Mesmaeker, 1993; De Mesmaeker, 1994A; De Mesmaeker, 1994B; Lebreton, 1993; Lebreton, 1994A; Lebreton, 1994B; Idsiak, 1993): Carbamate (Waldner, 1994; Stirchak, 1987; Habus, 1994; Thiocarbamate (Waldner,1995); Ureas (Waldner, 1994) Amines (De Mesmaeker,1994C; Caulfield, 1994); Hydroxylamine (Debart,1992; Vasseur,1992; Formacetal (Matteucci,1990; Jones, 1993)Thioformacetal (Jones,1993); Allyl ether (Cao,1994); Allyl, Ether, Thioether (Cao, 1994); Alkane (De Mesmaeker,1994; PNA A selection of binding and antisense properties (Nielsen, 1993A; Hanvey, 1992; Egholm, 1993; Nielsen, 1993B) ; PNA Synthesis (Egholm, 1992A; Egholm, 1992B);Prepn of purine PNA monomers and oligos (available commercially from Millipore corporation).

Modified bases may be synthesized as follows: Purine; synthesis and incorporation into ribozyme (Slim, 1992; Fu,1992; Fu, 1993); 7-deazaGuanosine, synthesis and incorporation into ribozyme (Fu, 1993); Inosine, synthesis and incorporation into ribozyme (Slim,1992; Fu, 1993)7-deazaAdenosine, synthesis and incorporation into ribozyme (Fu, 1992; Seela, 1993). O6-methylguanosine, synthesis and incorporation into ribozyme (Grasby, 1993); 2,6-diaminopurine, synthesis (Sproat, 1991); 2-aminopurine, synthesis and incorporation into ribozyme (Ng, 1994; Tuschl, 1993); Isoguanosine, synthesis and incorporation into ribozyme (Ng, 1994; Tuschl, 1993); Xanthosine, synthesis and incorporation into ribozyme (Tuschl, 1993);6-azathymidine, 6-aza-2'-deoxycytidine, synthesis and incorporation into oligonucleotides (Sanghvi, 1993); 5-alkenylpyrimidines; 5-propyne (Gilead, Froehler); inosine; 5-methylcytosine; pseudouridine; abasic ribose or deoxyribose.

SUMMARY OF NUCLEOTIDE-MODIFICATIONS WHICH HAVE BEEN TESTED IN RIBOZYMES

Sugars

Modifications may be made to the 2' OH group of the sugar at all non-conserved nucleotides; modifications tested have been 2' H (DNA), 2° F, 2' amino, 2'-O-allyl, 2'-O-methyl, 2'-C-allyl.

Selected modifications may be made to the 2' OH groups of the conserved nucleotides C3, U4, A6, N7, A9, G12, A13, A14, N15.2.

Modifications cannot be made to the 2' OH groups of GS, G8 and A15.1.

For a ribozyme with good cleavage activity, modifications should not be made to G5, A6, G8, G12, A15.1 (except G12 can be 2' H (DNA)).

Generally, except for modifications at G5, G8 and A15.1, no single modification causes a big reduction in cleavage activity; however, activity decreases as more modifications are included in the ribozyme.

Phosphates

The phosphate groups of the nonconserved nucleotides may be phosphorothioates (phosphorothioated DNA). Preferably, when non-conserved nucleotides are DNA, only two or three phosphates at the 3' and 5' ends of the ribozyme are phosphorothioates.

The phosphates 5' to the conserved nucleotides C3, U4, G5, G8 and G12, and 3' to A9 and N15.2, may be phosphorothioates; but phosphates 5' to A9, A13 and A14 may not be phosphorothioates.

Conserved nucleotides

C3

Sugar--2'-OH group can be modified (except, probably, for 2' amino). 2' H (Yang, 92), 2° F (Pieken, 91; Heidenreich, 92), 2'-O-allyl (Paolella, 92), 2'-O-Methyl (Usman, 95) are all modifications that permit cleavage.

Possibly cannot have 2' amino modification (several Cs in ribozyme had 2' amino modification which resulted in reduction in activity, and effect is probably due to 2' amino on C3 and/or C15.2) (Pieken, 91).

Phosphate--5' phosphate can be phosphorothioate (Shimayama, 93)

U4

Sugar--2' OH group can be modified. 2' H (Yang, 92), 2° F (Pieken, 91; Heidenreich, 92), 2' amino (Pieken, 91), 2'-C-allyl (Usman, 95), 2'-O-allyl (but keep as 2' OH if A6 is 2'-O-allyl) (Paolella, 92) are modifications that permit cleavage.

Phosphate--5' phosphate can be phosphorothioate (Shimayama, 93)

G5

Base--2-amino group on G base is essential (cannot be inosine) (Oddi, 90; Fu, 92)

Sugar--cannot make modifications to 2' OH of G5. Cannot have 2'H (Perreault, 90; Perreault, 91; Fu, 92; Williams, 92), 2' amino (Pieken, 91; Williams, 92), 2'-O-methyl (Paolella, 92), 2° F (Williams, 92).

A6

Base--can be purine (i.e. 6-amino group is not essential) (Fu, 92). N7 cannot be C7 in A base (Fu, 92).

Sugar--2' OH group can be modified. 2' H (Perreault, 90; Olsen, 91; Yang, 92; Fu, 92), 2° F (Olsen, 91), 2'-O-allyl (but only if U4 is 2' OH) (Paolella, 92) are modifications that permit cleavage.

N7

Seems to be a sensitive site for pyrimidine endonucleases; protection achieved if rN is rG or rA (Shimayama, 93).

Sugar--2' OH group can be modified. 2' H (tested dT) (Yang, 92), 2° F (Pieken, 91; Heidenreich, 92), 2'-amino (Pieken, 91), 2'-O-allyl (Paolella, 92), 2'-O-Methyl (Usman, 95) are all modifications that permit cleavage.

3' phosphate can be phosphorothioate (has been tested for N═U) (Shimayama, 93).

G8

Sugar--cannot make modifications to 2' OH of G8. Cannot have 2' H (Fu, 92; Williams, 92; Yang, 92), 2° F (Williams, 92), 2' amino (Williams, 92), 2'-O-allyl (Paolella, 92). (Perreault (91) says can have 2' H, but Yang (92) says this site is critical if lots of other conserved nucleotides are DNA.)

Phosphate--5' phosphate probably can be phosphorothioate (see N7 phosphate).

A9

Sugar--2' OH group can be modified. 2' H (Olsen, 91; Fu, 92; but Perreault (91) says cannot be 2' H), 2° F (Olsen, 91; Pieken, 91), 2'-O-allyl (Paolella, 92), 2'-O-Methyl (Usman, 95) are all modifications that permit cleavage.

Phosphate--5' phosphate cannot be phosphorothioate (Buzayan, 90; Ruffner, 90). 3' phosphate can be phosphorothioate (Shimayama, 93).

G12

Base--2-amino group is essential (cannot be inosine) (Slim, 92).

Sugar--2' OH group can tolerate some modifications. 2' H (Perreault, 91; Yang, 92; Williams, 92), 2' amino (Pieken, 91; Williams, 92) are OK. Cannot be 2° F (Williams, 92), 2'-O-allyl (Paolella, 92).

Phosphate--5' phosphate can be phosphorothioate (Shimayama, 93).

A13

Base--Can change N7 to C. in A base (Fu, 92). 6-amino group essential (cannot be purine) (Slim, 92).

Sugar--2' OH group can tolerate some modifications. 2' H (Perreault, 91; Yang, 92), 2'-O-allyl (Paolella, 92), 2'-O-Methyl (Usman, 95) are modifications that permit cleavage. Cannot have 2° F if each of A13, A14, A15.1 have 2° F (Pieken, 91).

Phosphate--5' phosphate cannot be phosphorothioate (Ruffner, 90).

A14

Base--Can change N7 to C. (Fu, 92). Can be purine (Slim, 92).

Sugar--2' OH group can tolerate some modifications. 2' H (Perreault, 91; Yang, 92), 2'-O-allyl (Paolella, 92), 2'-O-Methyl (Usman, 95) are modifications that permit cleavage. Cannot have 2° F if each of A13, A14, A15.1 have 2° F (Pieken, 91).

Phosphate--5' phosphate cannot be phosphorothioate (Ruffner, 90).

A15.1

Base--Can change N7 to C. (Fu, 92). 6-amino group essential (cannot be purine) (Slim, 92).

Sugar--Cannot modify 2' OH. Cannot have 2' H (Yang, 92), 2'-O-allyl (Paolella, 92), 2° F (if A13 and A14 also are 2° F) (Pieken, 91).

N15.2

Sugar--selected modifications permit cleavage. 2° F (Pieken, 91; Heidenreich, 92), 2'-O-allyl (Paolella, 92), 2'-O-Methyl (Usman, 95) are modifications that permit cleavage.

Rates are low if 2' H (Yang, 92). Possibly cannot have 2' amino modification (several Cs in ribozyme had 2' amino modification which resulted in reduction in activity, and effect is probably due to C3 and/or C15.2) (Pieken, 91). 3' phosphate can be phosphorothioate (Shimayama, 93)

Modifications at the 3' end of an oligonucleotide or ribozyme.

3' MEA(methoxyethylamine)phosphoramidate in last two (or last) internucleotide linkages; 3'--3' inverted diester linkage or 3'--3' inverted phosphoramidate (Shaw, 91). 3'--3' inverted Thymidine, or 3'--3' linked abasic ribose (Usman, 95).

Any combination of the above listed nucleotide modifications, substitutions, or derivatizations, made at the level of the sugar, base, or monophosphate groupings or phosphophodiester linkages may be made in the compounds provided that endonuclease activity is not lost.

The compounds of this invention may be incorporated and expressed in cells as a part of a DNA or RNA transfer vector, or a combination thereof, for the maintenance, replication and transcription of the ribozyme sequences of this invention.

Nucleotide sequences encoding the compounds of this invention may be integrated into the genome of a eukaryotic or prokaryotic host cell for subsequent expression (for example as described [Sambrook, 1989]). Genomic integration may be facilitated by transfer vectors which integrate into the host genome. Such vectors may include nucleotide sequences, for example of viral or regulatory origin, which facilitate genomic integration. Methods for the insertion of nucleotide sequences into a host genome are described for example in Sambrook et al. and Hogan et al. [Sambrook, 1989; Hogan, 1986; 1989].

Nucleic acid sequences encoding the ribozymes of this invention integrated into the genome may preferably include promoter and enhancer elements operably linked to the nucleotide sequence encoding the ribozyme of this invention, with an appropriate termination sequence and capable of expressing said ribozyme in a eukaryotic (such as animal or plant cells) or prokaryotic (such as bacteria) host cells. Ideally, the promoter and enhancer elements are designed for expression in a tissue and/or developmentally specific manner.

Additionally, the compounds of the present invention may be prepared by methods known per se in the art for the synthesis of RNA molecules. (For example, according to recommended protocols of Promega, Madison, Wis., USA). In particular, the ribozymes of the invention may be prepared from a corresponding DNA sequence (DNA which on transcription yields a ribozyme, and which may be synthesized according to methods known per se in the art for the synthesis of DNA) operably linked to an RNA polymerase promoter such as a promoter for T3 or T7 polymerase or SP6 RNA polymerase. A DNA sequence corresponding to a ribozyme of the present invention may be ligated into a DNA transfer vector, such as plasmid or bacteriophage DNA. Where the transfer vector contains an RNA polymerase promoter operably linked to DNA corresponding to a ribozyme, the ribozyme may be conveniently produced upon incubation with an RNA polymerase. Ribozymes may, therefore, be produced in vitro by incubation of RNA polymerase with an RNA polymerase promoter operably linked to DNA encoding a ribozyme, in the presence of ribonucleotides and an appropriate buffer. In vivo, prokaryotic or eukaryotic cells (including mammalian, plant and yeast cells) may be transfected with an appropriate transfer vector containing genetic material encoding a ribozyme in accordance with the present invention, operably linked to an RNA polymerase promoter such that the ribozyme is transcribed in the host cell. Transfer vectors may be bacterial plasmids or viral (RNA and DNA) or portion thereof. Nucleotide sequences corresponding to ribozymes are generally placed under the control of strong promoters such as, the lac, SV40 late, SV40 early, metallothionein, or lambda promoters. Particularly useful are promoters regulated in a tissue or a temporal (developmental) specific manner or tightly regulated inducible promoter suitable for gene theory, which may be under the control of exogenous chemicals. The vector may be an adenovirus or an adeno-associated virus. See for example PCT International Publication No. WO 93/03769, "Adenovirus Mediated Transfer of Genes to the Gastrointestinal Tract", U.S. Ser. No. 747,371; PCT International Publication No. WO 94/11506, "Adenovirus-Mediated Gene Transfer to Cardiac and Vascular Smooth Muscle," J. Leiden et al., U.S. Ser. No. 07/977,496; PCT International Publication No. WO 94/11522, "Vector for the Expression of Therapy-Relevant Genes," U. Stein et al.,PCT International Publication No. WO 94/11524, "Targetable Vector Particles," W. Anderson et al., U.S. Ser. No. 973,307;

PCT International Publication No. WO 94/17832, "Targeting and Delivery of Genes and Antiviral Agents into Cells by the Adenovirus Penton," G. Nemerow et al., U.S. Ser. Nos. 08/046,159 and 08/015,225. Ribozymes may be directly transcribed in vivo from a transfer vector, or alternatively, may be transcribed as part of a larger RNA molecule. For example, DNA corresponding to ribozyme sequences may be ligated into the 3' end of a reporter gene, for example, after a translation stop signal. Larger RNA molecules may help to stabilize the ribozyme molecules against nuclease digestion within cells. On translation the reporter gene may give rise to a protein, possibly an enzyme whose presence can be directly assayed.

The compounds of this invention may be involved in gene therapy techniques, where, for example, cells from a human suffering from a disease, such as HIV, are removed from a patient, treated with the ribozyme or transfer vector encoding the ribozyme to inactivate the infectious agent, and then returned to the patient to repopulateda target site with resistant cells, so called ex vivo therapy. In the case of HIV, nucleotide sequences encoding ribozymes of this invention capable of inactivating the HIV virus may be integrated into the genome of lymphocytes or may be expressed by a non-integrating vector such as adenovirus. Such cells would be resistant to HIV infection and the progeny thereof would also confer such resistance.

A transfer vector such as a bacterial plasmid or viral RNA or DNA or portion thereof, encoding one or more of the compounds, may be transfected into cells of an organism in vivo [See for example: Llewellyn, 1987; Hanahan, 1983]. Once inside the cell, the transfer vector in some cases may replicate and be transcribed by cellular polymerases to produce ribozyme RNAs which may have ribozyme sequences of this invention; the ribozyme RNAs produced may then inactivate a desired target RNA. Alternatively, a transfer vector containing one or more ribozyme sequences may be transfected into cells by electroporation, PEG, high velocity particle bombardment or lipofectants, or introduced into cells by way of micromanipulation techniques such as microinjection, such that the transfer vector or a part thereof becomes integrated into the genome of the host cell. Transcription of the integrated genetic material gives rise to ribozymes, which act to inactivate a desired target RNA.

Transfer vectors expressing ribozymes of this invention may be capable of replication in a host cell for stable expression of ribozyme sequences. Alternatively, transfer vectors encoding ribozyme sequences of this invention may be incapable of replication in host cells, and thus may result in transient expression of ribozyme sequences. Methods for the production of DNA and RNA transfer vectors, such as plasmids and viral constructs are well known in the art and are described for example by Sambrook et al. [Sambrook, 1989].

Transfer vectors would generally comprise the nucleotide sequence encoding the ribozyme of this invention, operably linked to a promoter and other regulatory sequences required for expression and optionally replication in prokaryotic and/or eukaryotic cells. Suitable promoters and regulatory sequences for transfer vector maintenance and expression in plant, animal, bacterial, and other cell types are well known in the art and are described for example in Hogan [Hogan, 1986; 1989].

The ribozymes of the present invention have extensive therapeutic and biological applications. For example, disease causing viruses in man and animals may be inactivated by administering to a subject infected with a virus, a ribozyme in accordance with the present invention adapted to hybridize to and cleave specific RNA transcripts of the virus. Such ribozymes may be delivered by parenteral or other means of administration. Alternatively, a subject infected with a disease causing virus may be administered a non-virulent virus such as vaccinia or adenovirus which has been genetically engineered to contain DNA corresponding to a ribozyme operably linked to an RNA promoter, such that the ribozyme is transcribed in the cells of the host animal, transfected with the engineered virus, to effect cleavage and/or inactivation of the target RNA transcript of the disease causing virus.

The ribozymes of the present invention have particular application to viral diseases caused for example, by the herpes simplex virus (HSV) or the AIDS virus (HIV). Further examples of human and animal disease which may be treated with the ribozymes of this invention include psoriasis, cervical preneoplasia, papilloma disease, bacterial and prokaryotic infection, viral infection and neoplastic conditions associated with the production of aberrant RNAs such as occurs in chronic myeloid leukemia. Diseases or infections which may be treated in plants with ribozymes of this invention include fungal infection, bacterial infections (such as Crown-Gall disease) and disease associated with plant viral infection. Of particular interest would be compounds targeting genes associated with male gametophyte development. Examples include PCT International Publication No. WO 92/18625, entitled "Male-Sterile Plants, Method For Obtaining Male-Sterile Plants And Recombinant DNA For Use Therein"; U.S. Pat. No. 5,254,802, entitled "Male Sterile Brassica Plants," S. Hoekstra et al.; PCT International Publication No. WO 93/25695, entitled "Maintenance of Male-Sterile Plants," M. Williams et al., claiming the priority of U.S. Ser. Nos. 07/970,840 and 07/899,072; PCT International Publication No. WO 94/25593, entitled "Method For Obtaining Male-Sterile Plants" Stiekema et al.; PCT International Publication No. WO 94/29465, entitled "Process For Generating Male Sterile Plants" Dirks et al.

The period of treatment would depend on the particular disease being treated and could be readily determined by a physician or by a plant biologist as appropriate. Generally treatment would continue until the disease being treated was ameliorated.

The ribozymes of the present invention also have particular application to the inactivation of RNA transcripts in bacteria and other prokaryotic cells, plants, animals and yeast cells. In bacteria, RNA transcripts of, for example, bacteriophage, (which cause bacterial cell death) may be inactivated by transfecting a cell with a DNA transfer vector which is capable of producing a ribozyme in accordance with the present invention which inactivates the phage RNA. Alternatively, the ribozyme itself may be added to and taken up by the bacterial cell to effect cleavage of the phage RNA. Similarly, eukaryotic and prokaryotic cells in culture may, for example, be protected from infection or disease associated with mycoplasma infection, phage infection, fungal infection and the like.

RNA transcripts in plants may be inactivated using ribozymes encoded by a transfer vector such as the Ti plasmid of Agrobacterium tumefaciens. When such vectors are transfected into a plant cell and integrated, the ribozymes are produced under the action of RNA polymerase and may effect cleavage of a specific target RNA sequence. Endogenous gene transcripts in plants, animals or other cell types may be inactivated using the compounds of the present invention. Accordingly, undesirable phenotypes or characteristics may be modulated. It may, for example, be possible using the ribozymes of the present invention to remove stones from fruit or treat diseases in humans which are caused by the production of a deleterious protein, or over production of a particular protein. The compounds described above may be used to effect male sterility by destroying the pollen production in a plant. Furthermore, for the in vivo applications of the ribozymes of this invention in humans, animals, plants, and eukaryotic and prokaryotic cells, such as in phenotypic modification and the treatment of disease, it is necessary to introduce the ribozyme into cells whereafter, cleavage of target RNAs takes place. In vivo applications are highly suitable to the compounds as discussed herein.

Methods for the introduction of RNA and DNA sequences into cells, and the expression of the same in prokaryotic and eukaryotic cells are well known in the art for example as discussed by Cotten and Friedman [Cotten, 1990; Friedman, 1989] . The same widely known methods may be utilized in the present invention.

The compounds of this invention may be incorporated into cells by direct cellular uptake, where the ribozymes of this invention would cross the cell membrane or cell wall from the extracellular environment. Agents may be employed to enhance cellular uptake, such as liposomes or lipophilic vehicles, cell permeability agents, such as dimethylsulfoxide, and the like.

The compounds of the present invention may be combined with pharmaceutically and veterinarally acceptable carriers and excipients which are well known in the art, and include carriers such as water, saline, dextrose and various sugar solutions, fatty acids, liposomes, oils, skin penetrating agents, gel forming agents and the like, as described for example in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., Easton, Pa., Edited by Ostol et al., which is incorporated herein by reference.

Agriculturally acceptable carriers and excipients are well known in the art and include water; surfactants; detergents; particularly biodegradable detergents; talc; inorganic and/or organic nutrient solutions; mineral earths and clays; calcium carbonate; gypsum; calcium sulfate; fertilizers such as ammonium sulfate, ammonium phosphate, urea, carborundum, and Agrobacterium tumefaciens; and natural products of vegetable origin such as, for example, grain, meals and flours, bark meals; and the like.

The compounds of this invention may be provided in a composition with one or more anti-viral, anti-fungal, anti-bacterial, anti-parasitic, anti-protazoan or antihelminthic agents, herbicides, pesticides or the like, for example as described in the Merck Index (1989) 11th Edition, Merck & Co. Inc.

By way of example only, therapeutic compositions of this invention may be directed against Herpes Simplex virus types 1 and 2, psoriasis, cervical preneoplasia, papilloma disease, and bacterial and prokaryotic infection. Such treatments may, for example, involve topical application of ribozyme to the site of disease. For example, in the treatment of Herpes virus lesions, ribozymes may be formulated into a cream containing a concentration of 0.1 nM to 100 mM ribozyme, preferably 1 nM to 1 mM. The cream may then be applied to the site of infection over a 1 to 14 day period in order to cause amelioration of symptoms of the infection. Prior to the final development of topical formulations for the treatment of virus infection, effectiveness and toxicity of the ribozymes and formulations involving them may, for example, be tested on an animal model, such as scarified mouse ear, to which virus particles, such as 2×10⁶ plaque forming units are added. A titer of infectious virus particles in the ear after treatment can then be determined to investigate effectiveness of treatment, amount of ribozyme required and like considerations. Similar investigations in animal models prior to human trials may also be conducted, for example, in respect of the treatment of psoriasis, papilloma disease, cervical preneoplasia, and in diseases such as HIV infection, bacterial or prokaryotic infection, viral infection and various neoplastic conditions, which neoplastic conditions involve a deleterious RNA species.

Compositions for topical application are generally in the form of creams, where the ribozymes of this invention may be mixed with viscous components. The compounds of this invention may be incorporated into liposomes or other barrier type preparations to shield the ribozymes from nuclease attack or other degradative agents (such as nucleases and adverse environmental conditions such as UV light).

Compositions may be provided as unit dosages, such as capsules (for example gelatin capsules), tablets, suppositories and the like. Injectable compositions may be in the form of sterile solutions of ribozyme in saline, dextrose or other media. Compositions for oral administration may be in the form of suspensions, solutions, syrups, capsules, tablets and the like. Ribozymes may also be provided in the form of an article for sustained release, impregnated bandages, patches and the like. The compounds of this invention may be embedded in liposomes or biodegradable polymers such as polylactic acid. Pharmaceutical compositions which may be used in this invention are described, for example, in Remington's Pharmaceutical Sciences, see above.

The present invention is further directed to a plant DNA expression cassette comprising a gene sequence flanked by promoter and terminator sequences at its 5'- and 3' ends respectively wherein said genetic sequence on expression provides a ribozyme RNA. The DNA cassette may further be part of a DNA transfer vector suitable for transferring the DNA cassette into a plant cell and insertion into a plant genome. In a most preferred embodiment of the present invention, the DNA cassette is carried by broad host range plasmid and which is capable of transformation into plant cells using Agrobacterium comprising Ti DNA on the left and right borders, a selectable marker for prokaryotes, a selectable marker for eukaryotes, a bacterial origin of replication and optional plant promoters and terminators such as pGA470. The present invention also includes other means of transfer such as genetic bullets (e.g. DNA-coated tungsten particles, high-velocity micro projectile bombardment) and electroporation amongst others [Maliga, 1993; Bryant, 1992; or Shimamoto, 1989].

The present invention is also directed to a transgenic plant resistant to a virus, its genome containing a sequence which gives rise, on transcription, to the nucleic acid molecule mentioned above. This transgenic plant, including fruits, and seeds thereof, may be from alfalfa, apple, arabidopsis, barley, bean, canola (oilseed rape), cantaloupe, carnation, cassava, casuarina, clover, corn, cotton, courgette, cucumber, eucalyptus, grape, melon, papaya, pepper, potato, rice, rose, snap dragon, soybean, squash, strawberry, sunflower, sweet pepper, tobacco, tomato, walnut, wheat or zucchini. Also included are the plant cells transformed by the above-mentioned transfer vector, as well as a prokaryotic, eukaryotic or yeast, plant or animal cell, comprising a nucleotide sequence which is, or on transcription gives rise to, the nucleic acid molecule.

The present invention will now be illustrated by way of non-limiting Examples only, with reference to the following non-limiting Examples, and Figures.

Example 1

Minizymes containing the deoxyribonucleotides d(GTTTT) and d(GTTTTT) between the conserved nucleotides A₉ and G₁₂ offer the following advantages:

(i) These minizymes show fast cleavage rates in vitro. See following data for the test systems Interleukin-2, TAT, CAT, and TNFα.

(ii) The CAT minizyme (CATMgtttt) shows activity against CAT in CHO cells (see Example 2).

(iii) The IL2 minizyme (IL2Mgtttt) shows activity against Interleukin-2 in PBMN cells (see Experiment 3) (Seq ID No. 5).

Minizyme g t t t t ##STR3## 1. Method for determining rates of cleavage (k_(obs)) by minizymes.

In these experiments, conditions are optimized so that the rate-limiting step in the reaction is cleavage of the substrate. The substrate consists of only a small number of nucleotides, in order to prevent strong self-association, and hence substrate association with the minizyme is not rate-limiting. In addition, the minizyme concentration is at least two-fold greater than substrate concentration, which is high enough to ensure all substrate molecules are bound by minizymes. Thus, the measured rate in these experiments should be rate of cleavage of the substrate.

The substrate is labelled on its 5' end with [³² p]-phosphate. In general, the minizyme and substrate are heated together in buffer for two minutes at 80° C. without magnesium, in order to denature the nucleic-acid molecules; however, this step has been shown not to be necessary in a number of cases. The cleavage reaction is initiated by adding Mg⁺⁺ to the mixture at 37° C. [MgCl₂ ]=10 mM, [Tris.HCl buffer]=50 mM, [Minizyme]=5 μM (typically), [Substrate]=2 μM (typically), temperature=37° C., pH 8.2 (for Interleukin-2 and TAT systems) and pH 8.0 (for CAT and TNFα systems). Samples are taken from the reaction mixture at various times, and the reaction is stopped by adding excess EDTA and formamide. The samples are electrophoresed on a polyacrylamide gel containing 7 M urea, and the amounts of 5'-product and uncleaved substrate are quantified using a PhosphorImager (Molecular Dynamics) and ImageQuant software. Kinetic parameters are obtained by fitting the data for % of product formed (P_(t)) versus time (t) to the equation

    P.sub.t =P.sub.28 -(exp(-k.sub.obs t)P.sub.Δ)

where P_(t) is the amount of product at time t, P.sub.∞ is the amount of product at t=∞, k_(obs) is the first-order rate-constant for the reaction, and P.sub.Δ is the difference between the percentage of product at t=∞ and t=0. This is a conventional first-order kinetic equation from which k_(obs), P.sub.∞, and P.sub.Δ are determined by least-squares fitting of the data.

2. Sequences of molecules.

Upper-case letters represent ribonucleotides, lower-case letters represent deoxyribonucleotides.

Interleukin-2 system (SEQ ID No. 6-10)

Substrate (15-mer)

5' UCCUGUC UUGCAUUg 3'

Minizyme g t t t t ##STR4##

Minizyme dg¹² dc¹⁵.2 ##STR5##

Minizyme c c t t ##STR6## Other interleukin-2 minizymes. "x" represents the deoxyribonucleotides forming the linker between the ribonucleotides. The various sequences of "x" that have been tested appear in the following table (Table 1). ##STR7## TAT system (Seq ID No. 11-13)

Substrate (13-mer) 5' GGAAGUC AGCCUa 3'

Minizyme g t t t t ##STR8##

Minizyme t t t t ##STR9## CAT system (Seq ID No.14-17)

Substrate (17-mer) 5' UUCCAUGUC GGCAGAAt 3'

Minizyme g t t t t ##STR10##

Minizyme t t t t ##STR11##

Minizyme g g t t t ##STR12## Tumor Necrosis Factor α(TNFα) system (Seq ID No. 18-20)

Substrate (20-mer) 5° CCAGGCAGUC AGAUCAUCUt 3'

Minizyme g t t t t ##STR13##

Minizyme t t t t ##STR14## 3. Observed cleavage rate (k_(obs) min⁻¹) and extent of cleavage (% P.sub.∞) of short substrates by minizymes with various linkers. "(Table 1B)"

Experimental conditions: 10 mM MgCl₂, 50 mM Tris.HCl, pH 8.2 (for Interleukin-2 and TAT systems) and pH 8.0 (for CAT and TNFα systems), 37° C., [Substrate]=2 μM, [Minizyme]=5 μM (except [TNFMtttt, TNFMgtttt]=4.3 μM).

    __________________________________________________________________________             Expt. 1 Expt. 2 Expt. 3                                                                               Mean                                            Minizyme linker                                                                        k.sub.obs                                                                          % P.sub.∞                                                                    k.sub.obs                                                                          % P.sub.∞                                                                    k.sub.obs                                                                          % P.sub.∞                                                                   k.sub.obs                                                                          (σ)                                                                           % P.sub.∞                                                                     (σ)                         __________________________________________________________________________     Interleukin-2                                                                    5' × 3'                                                                  c c t t 0.010 80#  0.011 80#    0.011 (.001)  80#                              t t t t   0.052 81.1 0.049 84.2 0.051 (.002) 83 (2)                            t t t t t   0.058 86.1 0.064 82.1 0.061 (.004) 84 (3)                          t t t t c 0.016 76.3 0.015 80.0   0.016 (.001) 78 (3)                          g t t t 0.118 82.3 0.107 84.5   0.113 (.008) 83 (2)                            g t t t t 0.299 83.5 0.287 85.3 0.225 84.9 0.270 (.040) 85 (1)                 g t t t t t 0.287 82.4 0.253 84.1   0.270 (.024) 83 (1)                        g t t t t t t 0.200 84.3 0.169 88.6   0.185 (.022) 86 (3)                      g t t t a 0.026 85.9 0.022 94.7   0.024 (.003) 90 (6)                          g t t t t a 0.047 81.0 0.038 88.4   0.043 (.006) 85 (5)                        g t t t g 0.122 82.3 0.092 89.8 0.107 83.9 0.107 (.015) 85 (4)                 g t t t t g 0.101 82.1 0.094 84.2   0.098 (.005) 83 (2)                        g g t t t 0.049 82.4 0.048 89.4   0.049 (.001) 86 (5)                          g t g t t 0.178 83.0 0.181 83.2   0.180 (.002) 83 (1)                          g t t g t 0.065 93.9 0.072 85.3   0.069 (.005) 90 (6)                          gtttt(dg.sup.12 dc.sup.15.2) 0.041 90.0 0.040 91.7   0.041 (.001) 91                                                      (1)                                 TAT                                                                            t t t t 0.074 89.1 0.067 91.9   0.071 (.005) 91 (2)                            g t t t t 0.179 89.7 0.190 90.8   0.185 (.008) 90 (1)                          CAT                                                                            t t t t 0.186 82.9 0.175 84.7   0.181 (.008) 84 (1)                            g t t t t    0.526* 60.8  0.448* 63.8  0.478* 62.8 0.48* (0.04) 62 (2)                                                     g t t t t    0.791* 72.0                                                      0.809* 71.4   0.80* (0.01)                                                     72 (1)                              g t t t t    0.650* 63.0                                                       g t t t t    0.453* 61.0                                                       g t t t t          0.59* (0.16) 65 (5)                                         g g t t t  0.278* 68.8  0.352* 67.6   0.32* (0.05) 68 (1)                      TNFα                                                                     t t t t 0.002  70.0# 0.002  70.0#   0.002 (.001)   70.0#                       g t t t t 0.274 59.0 0.180 74.2 0.303 58.7 0.25  (0.06) 64 (9)               __________________________________________________________________________      #fixed at this value.                                                           four different syntheses,   average of data for the four syntheses.           *reaction is biphasic; rate constant for the initial faster reaction is        given.                                                                   

                  TABLE 2                                                          ______________________________________                                         4. Minizymes with 5'd(GTTTT) linkers have improved cleavage                      activity in vitro compared with those with 5'd(TTTT) linkers.                  System     k.sub.obs (Mgtttt)                                                                        k.sub.obs (Mtttt)                                                                      k.sub.obs (Mgtttt)/k.sub.obs (Mtttt)           ______________________________________                                         Interleukin-2                                                                           0.270      0.051     5.3                                                TAT 0.185 0.071 2.6                                                            CAT 0.59  0.181 3.3                                                            TNFα 0.25  0.002 125                                                   ______________________________________                                    

The data in table 2 show that the minizymes with gtttt linkers consistently show k_(obs) values of 0.2 min⁻¹ or better. Since we know that IL2Mgtttt, with k_(obs) =0.27 min⁻¹, is active in cells (see Example 3), we can conclude that a minizyme with at least this level of activity in an in vitro system should not be hindered in cells by its k_(obs) value (i.e. rate of cleavage should not be rate limiting in cells), all other things being equal (such as target site being accessible).

Example 2. Minizyme Suppression of CAT Expression in CHO Cells.

Introduction

Minizymes are sequence specific RNA endonucleases derived from standard hammerhead ribozymes by elimination of helix II. Minizymes have been shown to exhibit significant in vitro cleavage activity against both short RNA targets as well as long transcribed RNA. This report describes the testing of a particular minizyme targeted against the mRNA of CAT (Chloramphenicol acetyl transferase) expressed in a mammalian cell line. The minizyme is a chimeric DNA/RNA molecule synthesized by solid phase methods and transfected into a CHO (chinese hamster ovary) cell line stably expressing CAT.

Experimental Protocol

A CHO based CAT expressing cell line MC 11, in which CAT is expressed from the Human metallothionein IIA (MT) promoter, was used in all experiments. The MT promoter is transcriptionally active at very low metal concentration and a reasonable level of CAT expression is observed in the absence of induction by added metals.

8×10⁴ cells were plated out in EMEM containing 10% foetal calf serum and allowed to attach overnight (14-16 hours). The cells were washed once with 1× PBS to remove serum, then the test molecules (pre-treated for 30 minutes with 1 μL of lipofectamine (GIBCO BRL, Life Technologies, Maryland, USA) in serum-free EMEM) were transferred to the cells. The final concentration of test molecules was 10 μM. After four hours both serum and Alamar Blue (Alamar Bio-Sciences Inc, Sacramento Calif.) (each to a final concentration of 10%) was added to the cells and incubation continued for a further 18 hours. At this time the supernatant was removed and the cumulative cell metabolic activity determined by measuring the extent of reduction of the Alamar Blue reagent. The cells were harvested and CAT activity assayed (Sleigh, 1986).

Alamar Blue Assay

Alamar Blue is a commercial material designed for use in cytotoxicity assays for cells in culture. The reagent is reduced intracellularly in an energy dependent fashion. The reduced form of the reagent is readily quantified by either its absorption spectrum or by fluorescence. We have quantified the reduced form of Alamar Blue by absorption spectroscopy.

Target mRNA

The target site in the CAT mRNA corresponds to CAT site 3 described in Haseloff and Gerlach (1988). The cleavage triplet is a GUC site and is located towards the 3' end 662 nucleotides from the ATG start codon.

Sequences of molecules

The test molecules are as follows (Seq ID No. 21-24):

Lower case letters are DNA, upper case letters are RNA.

    ______________________________________                                         tRNA      Yeast tRNA (Sigma) Phenol/Chloroform extracted.                      ______________________________________                                         N.sub.17  5' nnn nnn nnn nnn nnn nn 3' (n = a, g, c or t)                        CAT Antisense 5' att ctg ccg aca tgg aa 3'                                     CAT Minizyme 5' att ctg cc CUGAUGA gtttt GAAAC atg gaa 3'                      CAT Inactive 5' att ctg cc CUGAUGA gtttt GA GAC atg gaa 3'                     Minizyme                                                                     ______________________________________                                    

Bold G in CAT Inactive Minizyme represents the mutation that inactivates the minizyme.

Results

                  TABLE 3                                                          ______________________________________                                         Data from three Independent Experiments                                                               CAT Activity ±                                         Treatment Replicas SEM Alamar Blue ± SEM                                  ______________________________________                                         Experiment 1'                                                                    serum starved +                                                                            3        9385 ± 318                                                                            1.08 ± 0.06                                  lipofectamine                                                                  Minizyme 3 3788 ± 603  1.19 ± 0.06                                       Antisense 3 3885 ± 490 0.975 ± 0.05                                      Inactive 3 7043 ± 695 1.085 ± 0.01 (n = 2)                               Minizyme                                                                       N.sub.17 3 7254 ± 876 0.966 ± 0.06                                     Experiment 2'                                                                    serum starved +                                                                            2        10573 ± 455                                                                          1.045 ± 0.03                                  lipofectamine                                                                  tRNA 3 9680 ± 656 0.938 ± 0.04                                           Minizyme 3 4943 ± 974 0.953 ± 0.02                                       Antisense 3 5073 ± 467 0.883 ± 0.01                                      Inactive 3 6641 ± 642 0.872 ± 0.05                                       Minizyme                                                                       N.sub.17 3 4260 ± 342 0.857 ± 0.05                                     Experiment 3'                                                                    serum starved +                                                                            2        4143 ± 269                                                                            0.89 ± 0.05                                  lipofectamine                                                                  tRNA 3 5646 ± 235 0.781 ± 0.02                                           Minizyme 3 2855 ± 491 1.031 ± 0.07                                       Antisense 3 2978 ± 259 0.712 ± 0.10                                      Inactive 3 5969 ± 225 0.778 ± 0.04                                       Minizyme                                                                       N.sub.17 3 2210 ± 195 0.767 ± 0.04                                     ______________________________________                                    

                  TABLE 4                                                          ______________________________________                                         Mean ± SEM CAT Expression as a percentage of Control.                         (Controls are serum starved + lipofectamine treated cells)                                                           Mean ±                                Treatment Experiment 1 Experiment 2 Experiment 3 SEM                         ______________________________________                                         Control                                                                               100 ± 3.3                                                                               100 ± 4.3                                                                              100 ± 6.5                                                                           100                                        tRNA  91.6 ± 6.8 136.3 ± 4.2  114 ± 22                                Minizyme 40.4 ± 15.9  46.8 ± 19.7  68.9 ± 17.2 52 ± 9                                                   Antisense 41.4 ± 12.6 48.0 ±                                            9.2 71.9 ± 8.7 54 ± 9                                                     Inactive 75.0 ± 9.9  62.8 ±                                             9.7 144.1 ± 3.8   94 ± 25                                                 Minizyme                                  N.sub.17 77.3 ± 12.1 40.3 ± 8.0 53.3 ± 8.8  57 ± 11              ______________________________________                                    

FIG. 1 shows the CAT Expression (Mean±SEM) for Different treatments. Each experiment is performed in triplicate. FIG. 2 shows the extent of Alamar Blue Reduction (mean±SEM) in the same experiments as in FIG. 1. Extent of reduction relates to metabolic activity of the cells during the 18 hours post-transfection.

Discussion

The CAT minizyme contained the new linker 5' gtttt. When tested against a short 17-mer synthetic RNA substrate in vitro at 37° C., the minizyme cleaved the substrate with a reasonable rate constant (˜0.5 min⁻¹, t1/2˜1.4 minutes). In all experiments a constant number of cells (8×10⁴) were seeded and treated identically with the exception of the added oligonucleotides. Based on observed CAT activity, the random 17-mer does not appear to be an appropriate control molecule. Accordingly tRNA was included in two of the experiments to provide an alternative control.

The minizyme and the DNA antisense show similar levels of suppression (52±9%, 54±9%, respectively) and both are significantly more effective than the inactivated minizyme (94±25%, mean results Table 4). Thus both the minizyme and the antisense are showing activity in this cultured cell system.

We have been mindful of the danger of selecting a single protein or mRNA level as a specificity control and have therefore monitored the rate of general metabolism (by Alamar Blue reduction) as a measure of the specificity of the test molecules. It is interesting to note that the minizyme is apparently less toxic than the antisense in all experiments (Table 3, FIG. 2). Therefore the relative activity of the minizyme may be greater than that of the antisense, since a proportion of the apparent activity of the antisense molecule could result from a reduction in cell metabolism compared with minizyme treated cells.

Example 3

The activities of DNA-armed ribozymes and minizymes against Interleukin-2 mRNA in vivo.

The molecules tested

The following molecules (2-7) have been synthesized and tested for activity using one or more of the assays described below. Molecule 2 is a hammerhead ribozyme with deoxyribonucleotides in the arms which hybridize to the substrate. Molecule 3 is a minimized hammerhead ribozyme (minizyme) with a linker of sequence d(GTTTT) replacing stem-loop II of the full-sized ribozyme. Molecule 4 is a minizyme with 5' d(GTTTT) linker, which has been rendered inactive by replacing the conserved A₁₄ by a G. Molecule 5 is a DNA antisense control with sequence complementary to the 15-nucleotide target sequence. Molecule 6 is a DNA control with the same base composition as the DNA antisense, but with scrambled sequence. Molecule 1 is a 15-nucleotide, synthetic substrate with the same sequence as that targeted in the IL2 m-RNA; it is used to determine cleavage rate constants for the ribozyme and minizymes in vitro. In the sequences below, ribonucleotides are denoted by upper-case letters and deoxyribonucleotides by lower-case letters (Seq ID No. 25-31).

    ______________________________________                                         1.  5'  UCCUGUCUUGCAUUG   interleukin-2 target                                     sequence IL2S15                                                               - 2.                                                                                                    DNA-armed ribozyme  IL2R-DNA                          - 3.                                                                                                    minizyme with dGTTTT  linker IL2M.sub.GTTTT                                    - 4.                                                                           inactive minizyme  (GAAAC--GAGAC)  IL2M.sub.In                               active                                                  - 5. 3'  aggacagaacgtaac 5' DNA antisense                                       control IL2AS                                                                6. 3'  gaatcgcagaaagca 5' DNA random-                                            sequence control                                                               IL2C                                                                          - 7.                                                                                                    minizyme with  dCCTT linker  IL2M.sub.CCT          ______________________________________                                    

Preparation of oligonucleotides

The oligonucleotides were synthesised on an Applied Biosystems (Foster City, Calif.) model 391 synthesiser using protected DNA phosphoramidite monomers and RNA monomers, protected at the 2'-hydroxyl by tert-butyldimethylsilyl groups, from Millipore (Marlborough, Mass.). For convenience in the syntheses, the 3' nucleotide in all molecules is a deoxyribonucleotide. The methods used for the deprotection and gel-purification of the oligonucleotides were as described previously (McCall et al., 1992), with the exception that the oligonucleotides used for testing in cells were precipitated twice (rather than once) from 0.3 M sodium acetate and 80% ethanol, and then washed twice (rather than once) in cold 80% ethanol before drying under vacuum. The oligonucleotides were re-dissolved in autoclaved, de-ionized water. The concentrations of the oligonucleotide solutions were determined by UV spectroscopy using the following molar extinction coefficients for the nucleotides at 260 nm: A 15,400, G 11,700, C 7,300, T and U 8, 800 Lmol⁻¹ cm⁻¹. The purity of each oligonucleotide was checked by phosphorylating the 5' end using [γ-³² ]ATP and polynucleotide kinase (Boehringer Mannheim, Germany) as described previously (McCall et al., 1992). The oligonucleotides were stored at -20° C.

Determining rates of cleavage (k_(obs)) by minizymes

The rates at which the minizymes (IL2M_(GTTTT) and IL2M_(CCTT)) and ribozyme (IL2R-DNA) cleaved their cognate, short substrate (IL2S15) were determined in 10 mM MgCl₂, 50 mM Tris.HCl buffer, pH 8.25, at 37° C. with 5 μM minizyme and 2 μM substrate. The substrate was labelled on its 5' end with [32p]-phosphate. The minizyme and substrate were heated together in buffer for two minutes at 70° C., put to 37° C. for 2 minutes, centrifuged briefly at room temperature, and then returned to 37° C. for 2 minutes before starting the reaction by adding MgCl₂ (at 37° C.) to the mixture. The volume of the reaction mixture was generally 30 μL. 2 μL samples were taken at various times, and the reactions in these were quenched by adding 4 μL of 20 mM EDTA, 80% formamide, 0.1% bromphenol blue and 0.1% xylene cyanol. The samples were electrophoresed on a 15% polyacrylamide gel containing 7 M urea, and the amounts of 5'-product and uncleaved substrate were quantified using a Molecular Dynamics PhosphorImager (Sunnyvale, Calif.) and ImageQuant software. Kinetic parameters were obtained by fitting the data for % of product formed (P_(t)) versus time (t) to the equation

    P.sub.t =P.sub.∞ -(exp(-k.sub.obs t)P.sub.Δ)

where P_(t) is the amount of product at time t, P.sub.∞ is the amount of product at t=.sub.∞, k_(obs) is the first-order rate-constant for the reaction, and P.sub.Δ is the difference between the percentage of product at t=∞ and t=0. This is a conventional first-order kinetic equation from which k_(obs), P.sub.∞ and P.sub.Δ are determined by least-squares fitting of the data. The quoted rate constants are the mean (±standard deviation) of at least two experiments.

Assays for interleukin-2

Abbreviations: PBMN (peripheral blood mononuclear cells), PHA (phytohemagglutinin), IL-2 (interleukin-2).

Interleukin-2 is synthesised and secreted by T-lymphocytes following their activation by antigens or mitogens. In experiments to determine how well the test molecules could suppress the production of IL-2, an enriched population of human PBMN T-cells were transfected with the test molecules, and then stimulated with PHA to produce IL-2. In all cases, these test molecules were co-transfected with 1 μg tRNA and their effects on IL-2 levels were determined relative to the effect of the control which was 1 μg tRNA. The amounts of IL-2 secreted were assayed by three methods. In one method, IL-2 was assayed indirectly by measuring the ability of the supernatant from the PBMN cells to promote the growth of IL2-dependent mouse CTLL cells. In another, since IL-2 produced by PBMN T-cells promotes the growth of the same cells, IL-2 levels were assayed directly by measuring the growth of the stimulated cells. In addition to these bio-assays, immunoassays (which measure free and receptor-bound IL-2) were performed.

Indirect bio-assay

This assay is specific for interleukin-2 and is based on the absolute requirement of IL-2 for the growth of CTLL cells. Before use, the CTLL cells, maintained continuously proliferating in complete media containing added IL-2, were washed 3 times in complete media without added IL-2(Tables 5a and 5b).

In microtitre trays 100,000 PBMN cells (enriched in T-cells) were transfected in complete medium (RPMI plus 10% foetal-calf serum) with the test and control molecules at 5, 10 and 20 μM using 25 μg/mL DOTAP (Boehringer Mannheim, Germany) according to the manufacturer's instructions. After a period of 7 hours, the cells were stimulated with 5 μg/mL PHA (Sigma). After an additional 15-20 hours, supernatants from the PBMN cells were serially diluted and, in triplicate, 100 μL from each dilution were added to 5000 CTLL cells in 20 μL media. The CTLL cells were allowed to grow for 20 hours, were pulsed with ³ H-thymidine for 4 hours, were harvested, and then the DNA-associated radioactivity was determined. Slow growth of the CTLL cells (low level of incorporated ³ H-thymidine) indicates low levels of interleukin-2 in the supernatant, and hence a test molecule with good activity against interleukin-2 in the PBMN cells. The percentage inhibition of IL-2 by the test molecules is measured in the range where the cell growth-rate is linearly dependent on the amount of IL-2 (Sioud, 1994).

Direct bio-assay

PBMN cells were transfected with the test molecules for 12 hours, stimulated with PHA for about 48 hours, pulsed with ³ H-thymidine, and then harvested 18 hours later to determine DNA-associated radioactivity (Tables 6a and 6b).

Enzyme Amplified Sensitivity Immuno-Assay (EASIA)

Total IL-2 in the supernatant from PBMN cells transfected with 20 μM IL2M_(GTTTT) and 20 μM IL2M_(Inactive) was determined using IL2-EASIA, a solid-phase Enzyme Amplified Sensitivity Immuno-Assay performed in microtitre plates according to the manufacturer's instructions (MADGENIX) (Table 7).

Toxicity to cells

The viability of PBMN cells which had been transfected with test and control molecules was determined using acridine orange and ethidium bromide. Dead cells appear orange and live cells appear green in the presence of these indicators. The assays were performed on PBMN cells that were transfected with the test and control molecules for a period of 22-27 hours; these cells were not stimulated with PHA since this results in cell aggregation and hence inaccuracy in cell counting. In parallel, cells from the same donor were transfected with the test and control molecules and stimulated with PHA, and when supernatants from these cells were collected for assaying IL-2 levels, the viabilities of the cells not stimulated by PHA were determined.

Activities of DNA-armed ribozymes and minizymes

The original set of molecules synthesized for testing contained the DNA-armed ribozyme, the minizyme with d(CCTT) linker, the DNA antisense, and the DNA random-sequence control (molecules 2, 7, 5 and 6 respectively). In these first experiments using the indirect assay, with transfection concentrations of test molecules at 10 μM, the DNA-armed ribozyme showed good activity against IL2, while the minizyme with d(CCTT) linker and the DNA random-sequence control showed no activity; the DNA antisense showed about 50% the activity of the DNA-armed ribozyme. The activities of the DNA-armed ribozyme and the minizyme in cells correlated with the observed cleavage activities of these molecules as measured against the short synthetic IL2 substrate in vitro. Since the cleavage rate shown by the minizyme with d(CCTT) linker was extremely slow, we investigated the reason for this poor activity. In this investigation we found that by changing the linker from 5' d(CCTT) to 5' d(GTTTT) the cleavage rate of the minizyme could be increased 25-fold. The observed cleavage rate constants in 10 mM MgCl₂, at pH 8.25 and 37° C., for the DNA-armed ribozyme, the minizyme with d(CCTT) linker, and the new minizyme with d(GTTTT) linker, against their cognate, short substrate are 1.4 (0.2), 0.011 (0.001) and 0.27 (0.04) min⁻¹, respectively.

Following the discovery of the highly active minizyme with d(GTTTT) linker, we synthesized in large scale the DNA-armed ribozyme, the minizyme with d(GTTTT) linker, an inactivated minizyme with d(GTTTT) linker, the DNA antisense, and the DNA random-sequence control (molecules 2-6 above), and tested them for activity in cells.

Results from inpedendent experiments using molecules 2-6

In the tables below, the effectiveness of each test molecule (at different concentrations) in inhibiting the production of IL2 is presented as % inhibition averaged over two or three independent experiments (each done in duplicate or triplicate) with standard deviations given in brackets. In the first of each pair of tables, the data show the effectiveness of the test molecules against IL2, relative to that of the DNA random-sequence control taken as having 0% inhibitory effect. Since ribozymes and minizymes are being developed as alternatives to other oligonucleotide-based therapies, we believe that the inhibitory effects of these molecules, over and above the non-specific effects of a randomly-chosen oligonucleotide, are the data of interest. In most experiments, tRNA was also included as an additional control molecule. Generally, the DNA random-sequence control showed some activity against IL2 relative to tRNA, and so these data are presented for information (relative to tRNA taken as having 0% inhibitory effect) in the second of each pair of tables.

Indirect assay. % inhibition of IL2 as measured by the indirect assay.

                  TABLE 5A                                                         ______________________________________                                         Data relative to DNA random-sequence control.                                             Transfection concentration                                          Test molecule                                                                             5 μM     10 μM 20 μM                                       ______________________________________                                         IL2R-DNA   33          42 (13)  69 (1)                                           IL2M.sub.GTTTT 31 41 (4)   53 (11)                                             IL2M.sub.Inactive  5  6 (27) 28 (6)                                            IL2AS 10 21 (19) 13 (6)                                                      ______________________________________                                    

                  TABLE 5B                                                         ______________________________________                                         Data relative to tRNA.                                                                    Transfection concentration                                          Test molecule                                                                             5 μM     10 μM 20 μM                                       ______________________________________                                         IL2R-DNA   45          51 (9)   80 (4)                                           IL2M.sub.GTTTT 43 51 (3)  73 (12)                                              IL2M.sub.Inactive 22  22 (17)  47 (12)                                         IL2AS 25 35 (9)  49 (15)                                                       IL2C 18 16 (7) 35 (9)                                                        ______________________________________                                    

Direct assay. % inhibition of IL2 as measured in individual experiments by the direct assay.

                  TABLE 6A                                                         ______________________________________                                         Data relative to DNA random-sequence control.                                                   Transfection concentration                                      Test molecule 10 μM                                                       ______________________________________                                         IL2R-DNA     67 (3)                                                              IL2M.sub.GTTTT 46 (6)                                                          IL2M.sub.Inactive 24 (2)                                                       IL2AS  28 (14)                                                               ______________________________________                                    

                  TABLE 6B                                                         ______________________________________                                         Data relative to tRNA.                                                                          Transfection concentration                                      Test molecule 10 μM                                                       ______________________________________                                         IL2R-DNA     73 (4)                                                              IL2M.sub.GTTTT 55 (7)                                                          IL2M.sub.INactive 37 (2)                                                       IL2AS  40 (14)                                                                 IL2C 18 (4)                                                                  ______________________________________                                    

                  TABLE 7                                                          ______________________________________                                         EASIA                                                                            Transfection concentration 20 μM.                                             Test molecule                                                                               IU/ml  % inhibition relative to tRNA                          ______________________________________                                         IL2M.sub.GTTTT                                                                               70    72%                                                          IL2M.sub.Inactive 120 52%                                                      control (tRNA) 250 --                                                        ______________________________________                                    

Summary of results

The DNA-armed ribozyme (IL2R-DNA) and the minizyme with d(GTTTT) linker (IL2M_(GTTTT)) show activity specifically against interleukin-2 in human PBMN cells. The effects of these two molecules are greater than those measured for a DNA antisense molecule (IL2AS) and an inactivated minizyme (IL2M_(Inactive)) directed to the same target site on the IL2 mRNA, at transfection concentrations of 5, 10 and 20 μM. The molecules do not appear to be toxic to cells over a 24-hour period.

Example 4

Mini-ribozymes

Comments in publications on hammerhead ribozymes teach that helix II can be reduced to 2 b.p. without loss of activity, but further reduction to 1 b.p. results in at least a 10-fold reduction in activity (see data from Tuschl and Eckstein (1993) and from Long and Uhlenbeck (1994)). Note that these data are for all-RNA ribozymes. We have found that ribozymes with 1 b.p. in helix II, such that the sequence of "stem-loop II" is 5' GTTTC or 5' GTTTTC, where T may be dT or rU, have better than 10% the activity of analogous ribozymes with 4 b.p. in helix II. We call such ribozymes "mini-ribozymes". In some cases, the mini-ribozymes have observed cleavage rate constants which exceed that of the full-sized analogous ribozymes. Based upon published data, these findings are totally unexpected. Our data are presented below.

Sequences of mini-ribozymes and ribozymes

Upper-case letters represent ribonucleotides, and lower-case letters represent deoxyribonucleotides. Substrates are labelled S, followed by the number of nucleotides in the substrate molecule. Minizymes are labelled M, followed by the sequence of the linker connecting the conserved nucleotides A₉ and G₁₂ (e.g. KrMgttttc is a miniribozyme in the Kruppel system, with a deoxyribonucleotide linker of sequence 5' d(GTTTTC). Full-sized ribozymes which contain a stem-loop II (with 4 b.p. in helix II) are labeled RA (if made of ribonucleotides), RB (with deoxyribonuclotides in the arms which hybridize to the substrates, and ribonucleotides elsewhere), and RC (if made of deoxyribonuclotides, except for the conserved ribonucleotides C₃ -A₉ and G₁₂ -C₁₅.2). Interleukin-2 system (Seq ID No. 32-38) IL2S15 (15 mer substrate)

    5' UCCUGUCUUGCAUUg 3'

IL2Mgtttc (DNA/RNA mini-ribozyme with d(GTTTC) linker ##STR19##

IL2Mgttttc (DNA/RNA mini-ribozyme with d(GTTTTC) linker ##STR20##

IL2 "RB" DNA-armed ribozyme ##STR21##

IL2"RC" DNA-containing ribozyme ##STR22##

IL2 MGUUUUC(all-RNA miniribozyme with r(GUUUUC) linker ##STR23##

IL2 (all RNA ribozyme) ##STR24## TAT system (Seq. ID No. 39-43)

TATS13 (13 mer substrate)

    5' GGAAGUCAGCCU a 3'

TATS21 (21 mer substrate)

    5' UCCAGGAAGUCAGCCUAGGA c3'

TATMgtttc (DNA/RNA mini-ribozyme with d(GTTTC) linker ##STR25##

TATMgttttc (DNA/RNA mini-ribozyme with d(GTTTTC) linker ##STR26##

TAT"RC" DNA-containing ribozyme ##STR27## Kr system (Seq ID No. 44-47)

KrS13 (13 mer substrate)

    5' GCGAGUCCACAC t 3'

KrS21 (21 mer substrate)

    5' AUUUGCGAGUCCACACUGGA g 3'

KrMgttttc (DNA/RNA mini-ribozyme with d(GTTTTC) linker ##STR28##

Kr "RC" DNA-containing ribozyme ##STR29##

Experimental data

The method for determining rates of cleavage (kobs) of short substrates by the miniribozymes is as described in Example 1. Initial experiments at pH 8.2 showed the reactions for the TAT and IL2 miniribozymes and "RC" ribozymes are very fast, with the reactions being completed in less than 1 minute. Therefore, the reactions were performed at the lower pH of 7.1 (which in principle should be 12.6-fold slower than at pH 8.2 if the reactions have a first order dependence on OH concentration in this pH range). The data at pH 7.1 show more accurately the relative activities of the various molecules. In the Tables below, values in parentheses(σ) are the standard deviations of at least two independent determinations; and *indicates an initial fast reaction followed by a slower reaction, with kobs for the initial fast reaction being given.

                  TABLE 8                                                          ______________________________________                                         k.sub.obs (min.sup.-1) and % P.sub.∞ for KrMgttttc and KrRC              cleaving                                                                         the 13-mer and 21-mer substrates in 10 mM MgCl.sub.2, 50 mM Tris.Cl           pH 7.1, 37° C., 6 μM ribozyme/miniribozyme and 4 μM              substrate.                                                                            KrMgtttc         KrRC                                                   k.sub.obs (σ)                                                                          % P.sub.∞ (σ)                                                                k.sub.obs (σ)                                                                       % P.sub.∞ (σ)                   ______________________________________                                         KrS13  0.59* (0.09)                                                                              70(3)     1.6* (0.3)                                                                              78(2)                                       KrS21 3.0* (0.2) 58(1) 1.34* (0.08) 56(1)                                    ______________________________________                                    

                                      TABLE 9                                      __________________________________________________________________________     k.sub.obs (min.sup.-1) and % P.sub.∞ for TATMgtttc, TATMgttttc and       TATRC cleaving various                                                           substrates in 10 mM MgCl.sub.2, 50 mM Tris.Cl pH 7.1, 37° C.          Concentration of                                                                 ribozyme exceeded that of substrate, with concentrations varying             between 4 μM and 8 μM                                                      (ribozyme and miniribozyme) and 2 μM and 5 μM (substrate), with        typical concentrations                                                           being 6 μM for ribozyme/miniribozyme and 4 μM for substrate.                TATMgtttc  TATMgttttc  TATRC                                              k.sub.obs (σ)                                                                       % P.sub.∞ (σ)                                                           k.sub.obs (σ)                                                                   % P.sub.∞ (σ)                                                           k.sub.obs (σ)                                                                  % P.sub.∞ (σ)                    __________________________________________________________________________     TATS13                                                                              0.06 (0.02)                                                                          78 (6)                                                                              0.175* (0.003)                                                                        74 (7)                                                                              0.43* (0.08)                                                                         80 (7)                                         TATS21 1.1* (0.3)  39 (1) 0.9* (0.2) 45 (3) 0.45* (0.09) 31 (2)              __________________________________________________________________________

                  TABLE 10                                                         ______________________________________                                         k.sub.obs (min.sup.-1) and % P.sub.∞ for the DNA-containing              IL2Mgtttt,                                                                       IL2Mgttttc, IL2Mgtttc, IL2RB and IL2RC, and the all-RNA                        IL2MGUUUUC and IL2RA cleaving the substrate IL2S15 in 10 mM                    MgCl.sub.2, 50 mM Tris.Cl pH 7.1, 37° C. Concentration of             ribozyme                                                                         exceeded that of substrate, with concentrations varying between               2.5 μM and 20 μM (ribozyme), and 1 μM and 8 μM (substrate,        with                                                                             typical concentrations being 5 μM for ribozyme/miniribozyme and            2 μM for substrate.                                                                       k.sub.obs (σ)                                                                       % P.sub.∞ (σ)                            ______________________________________                                         IL2Mgtttc      0.119  (0.004) 85(4)                                              IL2Mgttttc 0.19 (0.02) 82(1)                                                   IL2RB 0.06 (0.02) 78(5)                                                        IL2RC 0.05 (0.01) 90(7)                                                        IL2MGUUUUC 0.316 (0.001) 76(2)                                                 IL2RA 1.4* (0.2) 66(1)                                                       ______________________________________                                    

                  TABLE 11                                                         ______________________________________                                         Comparing the rates of cleavage of substrates (at                                37° C., pH 7.1, 10 mM MgCl.sub.2) by the miniribozymes with           d(GTTTC)                                                                         and d(GTTTTC) linkers relative to the analogous DNA-containing                 ribozymes RC, and the all-RNA mini-ribozyme with r(GUUUUC)                     linker relative to the analogous all RNA ribozyme RA.                                 k.sub.obs gtttc/RC                                                                        k.sub.obs gttttc/RC                                                                       k.sub.obs GUUUUC/RA                             ______________________________________                                         KrS13    --         0.37       --                                                KrS21 -- 2.2 --                                                                TATS13  0.14 0.41 --                                                           TATS21 2.4 2.0 --                                                              IL2S15 2.4 3.8 0.23                                                          ______________________________________                                    

Comments

Several of the molecules used (Kr"RC", TAT"RC", IL2S15, IL2Mgtttc and IL2"RC") have been synthesized at least twice. The rates of cleavage and extents of cleavage do not vary significantly for molecules from different syntheses.

The IL2 minizyme with d(GTTTT) linker, which has kobs=0.27 min⁻¹ at pH 8.2 (and kobs=0.024 (0.005) min⁻¹ at pH 7.1), is active in cells (see Example 3). Therefore, the IL2 mini-ribozymes with d(GTTTC) and d(GTTTTC) linkers, which have kobs=0.11 and 0.19 min⁻¹, respectively, at pH 7.1, and which target the same site in the IL2 mRNA, are expected to be active in cells. For similar reasons, the all-RNA IL2 mini-ribozyme IL2MGUUUUC, which has kobs=0.316 min⁻¹ at pH 7.1, is expected to be active in cells.

References

Altman, S. (1987) Adv. Enzymol. 62:1.

Aurup, H., Williams, D. M. & Eckstein, F. (1992), Biochem. 31:9636-9641.

Augustyns, K., F. Vandendriessche, A. A. Van, R. Busson, C. Urbanke and P. Herdewijn. (1992), Nucleic Acids Res. 20:4711-6.

Baer, M. F., et al. (1990), Methods in Enzymology, 181:569.

Beaton, G., Dellinger, D., Marshal, W. S. and Caruthers M. H. "Synthesis of oligonucleotide phosphorodithioates" in Oligonucleotides and Analogues; a Practical Approach pp 109-136, F. Eckstein (Ed). Oxford University Press, Oxford.

Beijer, B., Grotli, M., Douglas, M. E. and Sproat, B. S. (1994), Nucleos Nucleot., 13:1905-1927.

Blommers, M. J., U. Pieles and A. De Mesmaeker (1994), Nucleic Acids Res., 22:4187-4194.

Breaker, R. R. and Joyce, G. F. (1994), Tibtech, 12:268-275.

Bruening, G., (1989), Methods in Enzymology, 180:546-558.

Bruening, G. (1990), Seminars in Virol., 1:127.

Bryant, J. (1992), Tibtech, 10:342-343.

Buzayan et al. (1986), Proc. Natl. Acad. Sci. USA, 83:8859-8862.

Buzayan, J. M., van Tol, H., Feldstein, P. A. and Breuning, G. (1990), Nucleic Acids Res., 18: 4447-4451. Phosphate 5' to A9 cannot be phosphorothioate.

Cameron et al. (1989), Proc. Natl. Acad. Sci. USA, 86:9139-9143.

Cantor, G. H. et al. (1993), Proc. Natl. Acad. Sci. USA, 90:10932-10936.

Cao, X. and M. D. Matteucci (1994), Tet. Lett., 35:2325-2328.

Carruthers et al. (1987), Methods in Enzymology, 154: 287-313).

Caruthers, M. H. et al (1991), Nucleic Acids Symp Ser. (ENGLAND), 24:91-94.

Caulfield, T. J., Prasad, C. V. C., Prouty, C. P., Saha, A. K., Sardaro, M. P., Schairer, W. C., Yawman, A., Upson, D. A., Kruse, L. I. (1994), Bioorg. Med. Chem. Lett., 3:2771-2776.

Cech, T. R. (1987), Science, 236:1532-1539.

Cech, T. R. et al. (1981), Cell, 27:487.

Chen, J. K., R. G. Schultz, D. H. Lloyd and S. M. Gryaznov (1995), Nucleic Acids Res., 23(14):2661-2668.

Chur, A., B. Holst, O. Dahl, H. P. Valentin and E. B. Pedersen (1993), Nucleic Acids Res., 21(22):5179-83.

Conrad, F. et al. (1995), Nucleic Acids Res., 23:1845-1853.

Cotten, M. (1990), Tibtech, 8:174-178.

Cotten, M. et al. (1989), The EMBO Journal, 8:3861-3866.

Debart, F., Rayner, B., Degols, G., & Imbach, J-L. (1992A), Nucleic Acids Res., 20:1193-1200.

Debart, F., J. J. Vasseur, Y. S. Sanghvi and P. D. Cook. (1992B), Bioorg. Med. Chem. Lett., 2:1479-1482.

De Mesmaeker, A., J. Lebreton, A. Waldner, V. Frisch, R. M. Wolf and S. M. Freier (1993), Synlett, 733-736.

De Mesmaeker, A., J. Lebreton, A. Waldner, V. Frisch and R. M. Wolf. (1994A), Bioorg. Med. Chem. Lett., 4:873-878.

De Mesmaeker, A., A. Waldner, J. Lebreton, P. Hoffmann, V. Frisch, R. M. Wolf and S. M. Freier. (1994B), Angew. Chem. Int. Ed., 33:226-229.

De Mesmaeker, A., A. Waldner, Y. S. Sangvhi and J. Lebreton. (1994C), Bioorg. Med. Chem. Lett., 4:395-398.

De Mesmaeker, A., A. Waldner, Y. S. Sangvhi and J. Lebreton. (1994), Bioorg. Med. Chem. Lett., 4:395-398.

De Mesmaeker, A., Altmann, K. H., Waldner, A., Wenderborn, S. (1995), Current Opinion in Structural Biology, 5:343-355.

Egholm, M., O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden and P. E. Nielsen. (1993), Nature, 365:566-8.

Egholm, M., O. Buchardt, P. E. Nielsen and R. H. Berg. (1992A), J. Am. Chem. Soc., 114:1895-1897.

Egholm, M., P. E. Nielsen, O. Buchardt and R. H. Berg. (1992B), J. Am. Chem. Soc., 114:9677-9678.

Eldrup, A. B., K. Bjergarde, J. Felding, J. Kehler and O. Dahl. (1994), Nucleic Acids Res., 2296(1082):1797-1804.

Epstein, L. M., and Gall, J. G., (1987) Cell 48:535.

Epstein, L. M., and Gall, J. G., (1989) Cold Spring Harbour Symp. Ouant. Biol. 52:261.

Evans, G. J., et al. (1992) Biochem. Soc. Trans. 20:344S.

Fathi, R., Q. Huang, G. Coppola, W. Delaney, R. Teasdale, A. M. Krieg and A. F. Cook. (1994A), Nucleic Acids Res. 22(24). 5416-5424.

Fathi, R., Q. Huang, J. L. Syi, W. Delaney and A. F. Cook. (1994B), Bioconjug. Chem. 5(1). 47-57.

Foehler et al. (1986) Nucleic Acids Res. 14: 5399-407).

Friedman, T., (1989) Science 244: 1275-1280.

Froehler, B., P. Ng and M. Matteucci. (1988), Nucleic Acids Res., 16(11). 4831-9.

Fu, D. J. and L. W. McLaughlin. (1992A), Proc. Natl. Acad. Sci. USA. 89(9). 3985-9.(G5, G8 can't be DNA. A6 and A9 can be DNA. G5 can't be inosine. G8 can be inosine, and A6 and A9 can be purine.)

Fu, D. -J. and McLaughlin, L. W. (1992B), Biochemistry, 31, 10941-10949. (Can't change atom N(7) to C(7) in A6; but can change N(7) to C(7) in A9, A13, A14, A15.1.)

Fu, D. -J., S. B. Rajur and L. W. McLaughlin. (1993), Biochemistry. 32 10629-10637.

Fu, D. -J. and L. W. McLaughlin. (1992), Biochemistry. 31 10941-10949.

Garbesi, A., M. L. Capobianco, F. P. Colonna, L. Tondelli, F. Arcamone, G. Manzini, C. W. Hilbers, J. M. Aelen and M. J. Blommers. (1993), Nucleic Acids Res. 21, 4159-65.

Goodchild, J. (1992), Nucleic Acids Res. 20, 4607-4612. (2'-O-methylation in hybridizing arms enhances cleavage and improves resistance to nucleases.)

Grasby, J. A., P. Jonathan, G. Butler and M. J. Gait. (1993), Nucleic Acids Res. 21(19). 4444-50.

Gryaznov, S. M. and R. L. Letsinger. (1992A), Nucleic Acids Res. 20(13). 3403-9.

Gryaznov, S. M. and R. L. Letsinger. (1992B), Nucleic Acids Res. 20(13). 3403-9.

Gryaznov, S. M., D. H. Lloyd, J. K. Chen, R. G. Schultz, L. A. DeDionisio, L. Ratmeyer and W. D. Wilson. (1995), Proc. Natl. Acad. Sci. USA 92(13). 5798-5802.

Green, R., and Szostak, J. W. (1992) Science 258:1910.

Habus, I., J. Temsamani and S. Agrawal. (1994), Biorg. Med. Chem. Lett. 4 1065-1070.

Hanahan et al., (1983), J. Mol. Biol. 166).

Hanvey, J. C., N. J. Peffer, J. E. Bisi, S. A. Thomson, R. Cadilla, J. A. Josey, D. J. Ricca, C. F. Hassman, M. A.

Bonham, K. G. Au and a. 1. et. (1992), Science. 258(5087). 1481-5.

Haseloff, J. and W. L. Gerlach, (1988) Nature, 334:585-591.

Heidenreich, O. and Eckstein, F. (1992), J. Biol. Chem., 267, 1904-1909. (2'-fluorocytidine substitutions have no effect on cleavage rates, but 2'-fluorouridine substitutions cause 5-fold decrease in k_(cat) /K_(M). One 5' terminal and three 3' terminal phosphorothioate groups have little effect on rates. Putting all pyrimidines as 2'-fluoropyrimidines, and using the one 5' terminal and three 3' terminal phosphorothioate groups, gave a 7-fold reduction in k_(cat) /R_(M), and 50-fold improvement in stability.)

Heidenreich, O., Benseler, F., Fahrenholz, A. and Eckstein, F. (1994), J. Biol. Chem. 269, 2131-2138.

Hendry, P. et al (1994), Biochim et Biophys Acta, 1219: 405-412.

Herschlag, D., and Cech, T. R. (1990) Nature 344:405.

Hogan, B. et al., (1986), Manipulating the Mouse Embryo, A Laboratory Manual, Cold Spring Harbor.

Hogan, B. et al., (1989), Science, 244: 1275.

Homann, M., et al. (1993) iNucleic Acid Res. 28:2809-2814.

Idsiak, I., G. Just, M. J. Damha and P. A. Gianaris. (1993), Tet. Lett. 34 5417-5420.

Jager, A., M. J. Levy and S. M. Hecht. (1988), Biochemistry. 27(19). 7237-46.

Jones, R. J., K. Y. Lin, J. F. Milligan, S. Wadwani and M. D. Matteucci. (1993), J. Org. Chem. 58 2983-2991.

Kruger, K., et al. (1982) Cell 31:147.

Lamb, J. W. & Hay, R. T., (1990) J. Gen. Virol., 71:2257-2264.

Lebreton, J., A. De Mesmaeker, A. Waldner, V. Fritsch, R. Wolf and S. M. Freier. (1993), Tet. Lett. 34 6383-6386.

Lebreton, J., A. Waldner, C. Lesueur and A. De Mesmaeker. (1994A), Synlett, 137-140.

Lebreton, J., A. Waldner, V. Fritsch, R. M. Wolf and A. De Mesmaeker. (1994B), Tet. Lett. 35 5223-5228.

Letsinger, R. L., Singman, C. N., Histand, G., Salunkhe, M. (1988), J. Am. Chem. Soc., 110, 4470-4471.

L'Huillier, P. J. L., et al. (1992) The EMBO Journal 11:4411-4418.

Llewellyn et al., (1987), J. Mol. Biol., 195: 115-123.

Long, D. M. & Uhlenbeck, O. C., (1994), Proc. Natl. Acad. Sci. USA 91:6977-6981.

Maliga, P. (1993) Tibtech 11:101-106.

Matteucci, M. D. (1990), Tet. Lett. 31:2385-2388.

Mazzolini, L., et al. (1992) Plant Molecular Biology 20:715-731.

McCall, M. J., Hendry, P. and Jennings, P. A. (1992) Proc. Natl. Acad. Sci. USA, 89, 5710-5714.

Miller P. S., Bahn, P., Cushman, C D., Kean, J. M., Levis, J. T. (1991), Nucleosides and Nucleotides, 10, 27-46.

Monia, B. P., E. A. Lesnik, C. Gonzalez, W. F. Lima, D. McGee, C. J. Guinosso, A. M. Kawasaki, P. D. Cook and S. M. Freier. (1993), J. Biol. Chem. 268(19). 14514-22.

Ng, M. M. P., F. Benseler, T. Tuschl and F. Eckstein. (1994), Biochemistry 33(40). 12119-12126.

Nichols, M., et al. (1988) Proc. Natl. Acad. Sci. USA 85:1379.

Nielsen, P. E., M. Egholm, R. H. Berg and O. Buchardt. (1993A), Anticancer Drug Des. 8(1). 53-63.

Nielsen, P. E., M. Egholm, R. H. Berg and O. Buchardt. (1993B), Nucleic Acids Res. 21(2). 197-200.

Noller, H. F., et al. (1992) Science 256:1416.

Odai, O., Hiroaki, H., Sakata, T., Tanaka, T. and Uesugi, S. (1990), FEBS Lett. 267, 150-152. (G5 cannot be inosine; the 2-amino group seems to be important for stability.)

Olsen, D. B., F. Benseler, H. Aurup, W. A. Picken and F. Eckstein. (1991), Biochemistry 30 9735-9741.(Single 2'-fluoro or 2'-H (DNA) substitutions on adenosines have little effect on cleavage, but results are additive, so that cleavage drops as more modifications are added. 2' deoxy or 2'-fluoro modifications at A6 and A9 are OK; A13-A15.1 are more sensitive.)

Pace, N. R., and Smith, D., (1990) J. Biol. Chem. 265:3587.

Pan, T., and Uhlenbeck, O. C. (1992) Nature 358:560.

Paolella, G., Sproat, B. S. and Lamond, A. I. (1992), The EMBO Journal 11, 1913-1919. (Can make all nucleotides 2'-O-allyl ribonucleotides except U4, G5, A6, G8, G12, A15.1. Of these six ribonucleotides, either U4 or A6, but not both, may be 2'-O-allyl, without much loss in activity, but better activity is achieved if U4 and A6 are unmodified RNA. With U4, G5, A6, G8 and A15.1 as RNA and other nucleotides 2'-O-allyl, G12 can be DNA.)

Perreault, J. -P., Wu, T., Cousineau, B., Ogilvie, K. K. and Cedergren, R. (1990), Nature, 344, 565-567. (Putting DNA into conserved nucleotides A6, G8, A9, G12, A13 and A14 reduces activity, but doesn't eliminate it. Making G5 as DNA results in big reduction in activity.)

Perreault, J. -P., Labuda, D., Usman, N., Yang, J. -H. and Cedergren, R. (1991), Biochemistry, 30, 4020-4025. (G5 and A9 cannot be DNA. G12, A13, A14, G8 can be DNA.)

Perriman, R., et al. (1993) Antisense Res. & Dev. 3:253-263.

Piccirilli, J. A., et al. (1992) Science 256:1420.

Pieken, W. A., Olsen, D. B., Aurup, H., Williams, D. M., Heidenreich, O., Benseler, F., and Eckstein, F. (1991), Nucleic Acids Res. Symposium Series, 24. (2' amino group at C15.2 (together with 3 other 2'-amino Cs in hybridizing arms) has reduction in activity. Can't have 2'-amino at G5, but can at G12.)

Pieken, W. A., Olsen, D. B., Benseler, F., Aurup, H. and Eckstein, F. (1991), Science, 253, 314-317. (Can make all cytidines (C3, C15.2) and uridines (U4, U7) 2'-fluoro or 2'-amino and not get much reduction in cleavage activity. A9 can be 2'-fluoro, but A13, A14 and A15 together cannot have 2'-fluoro group.)

Pyle, A. M. (1993) Science 261:709-714.

Robertson, D. L., and Joyce, G. F. (1990) Nature 344:467.

Ruffner, D. E. and Uhlenbeck, O. C. (1990) Nucleic Acids Res., 18: 6025-6029.

Sambrook, J. et al., (1989), Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press.

Saenger, W. (1984), Principles of Nucleic Acid Structure, Springer-Verlag, N.Y.

Sanghvi, Y. S., G. D. Hoke, S. M. Frier, M. C. Zounes, C. Gonzalez, L. Cummins, H. Sasmor and P. D. Cook. (1993). Nucleic Acids Res. 21 3197-3202.

Saville, B. J. and Collins, R. A. (1990) Cell 61:685.

Saxena, S. et al., (1990) J. Biol. Chem., 265:17106-17109.

Seela, F., K. Mersmann, J. A. Grasby and M. J. Gait. (1993). Helv. Chim. Acta. 76 1809-1820.

Shaw, J. -P., Kent, K., Bird, J., Fishback, J. and Froehler, B. (1991) Nucleic Acids Res., 19, 747-750. (Degradation of oligodeoxynucleotides in fetal calf serum and cell supernatant is predominantly by 3' exonucleases. Good protection from these 3' exonucleases (and little change in melting temperatures of duplexes) is achieved by synthesizing oligodeoxyribonucleotides with one or two methoxyethyleneamine(MEA) phosphoroamidate substitutions at the 3' end, or by an inverted linkage (3'--3') using an inverted diester or an inverted phosphoroamidate. Uniformly substituted phosphorothioate oligonucleotides were also very stable to 3' exonucleases, but melting temperatures of duplexes were lowered by 10° C.)

Shimamoto, K., Terada, R., Izawa, T., and Fujimoto, H. (1989) Nature 338:274-276.

Shimayama, T., Nishikawa, F., Nishikawa, S. and Taira, K. (1993) Nucleic Acids Res. 21, 2605-2611. (Most stable and active Rz had phosphorothioate-DNA in stems I, II and III, as well as thio on 3' side of rC3, rU4, rA9, rC15.2; rN7 is rA or rG (not thio). Protocol given for synthesis and workup of chimeric thio-DNA/RNA ribozymes.)

Sinha, N. D., Biernat, J, & Koster, H. (1984) Nucleic Acids Res. 12, 4539-4557.

Sioud, M. and Drlica, K. (1991) Proc. Natl. Acad. Sci. USA 88:7303-7307.

Sioud, M. (1994) J. Mol. Biol., 242, 619-629.

Slim, G. and M. Gait. (1992). Biochem. Biophys. Res. Commun. 183 605-609.(G12 cannot be inosine; A13, A15.1 cannot be purine. A14 can be purine. G8 can be inosine (but rates down 10-fold) and A9 can be purine (but rates down 6-fold).)

Sleigh, M. J. Anal. Biochem. 156, 251-256 (1986)

Strobel, S. A. et al., (1991), Nature 350: 172-174 and references therein.

Sober, H. (1970), CRC Handbook of Biochemistry, Second edition.

Sproat et al. Oliconucleotide Synthesis--A Practical Approach, IRL Press, Oxford (1984) M. J. Gait--Editor, pp. 83-115).

Sproat, B. S. and Lamond, A. I. (1991A) 2'-O-methyloligoribonucleotides: Synthesis and applications. in Oliconucleotides and Analogues; a Practical Approach pp 49-86,, F. Eckstein (Ed). Oxford University Press, Oxford.

Sproat, B. S., A. I. Lamond, R. G. Garcia, B. Beijer, U. Pieles, M. Douglas, K. Bohmann, F. M. Carmo, S. Weston and S. O'Loughlin. (1991B). Nucleic Acids Symp Ser. 1991 (24). 59-62.

Stec, W. J.; Grajkowski, A.; Koziolkiewicz, M.; Uznanski, B. (1992). Nucleic Acids Res. 19, 5883-8

Stirchak, E. P., J. E. Summerton and D. D. Weller. (1987). J. Org. Chem. 52 4202-4206.

Sullenger, B. A. and Cech, T. R. (1993) Science 262:1566-1569.

Summers, M. F., C. Powell, W. Egan, R. A. Byrd, W. D. Wilson and G. Zon. (1986). Nucleic Acids Res. 14(18). 7421-36.

Symons, R. H., (1989) TIBS, 14:445-450.

Symons, R. H., (1990) Seminars in Virol. 1:117.

Szabo, T., A. Kers and J. Stawinski. (1995). Nucleic Acids Res. 2380(60204). 893-900.

Szostak, J. W. (1992) TIBS, 17:89-93.

Taylor, J., (1990) Seminars in Virol. 1:135.

Tuschl, T. & Eckstein, F., (1993), Proc. Nat. Acad. Sci. USA, 90:6991-6994.

Uhlmann et al., (1990), Chem. Revs., 90:544-584.

Tuschl, T., M. M. P. Ng, W. Pieken, F. Benseler and F. Eckstein. (1993). Biochemistry. 32(43). 11658-11668.

Usman, N., Beigelman, L., Draper, K., Wincott, F and McSwiggen, J. (1995) RNA synthesis and ribozymes. J. Cellular Biochemistry, Absract supplement 19A, Abstract A6-018, p 205.

Vasseur, J. J., F. Debart, Y. S. Sanghvi and P. D. Cook. (1992). J. Am. Chem. Soc. 114 4006-4007.

Vyazovkina, E. V., E. V. Savchenko, S. G. Lokhov, J. W. Engels, E. Wickstrom and A. V. Lebedev. (1994). Nucleic Acids Res. 2202(1287). 2404-2409.

Waldner, A., A. De Mesmaeker and J. Lebreton. (1994A). Bioorg. Med. Chem. Lett. 4 405-408.

Waldner, A. and A. De Mesmaeker. (1995). Synlett. 108-110.

Waldner, A., A. De Mesmaeker, J. Lebreton, V. Fritsch and R. M. Wolf. (1994B). Synlett. 57-61.

Waugh, D. S., et al. (1989) Science 244:1569.

Williams, D. M., Pieken, W. A. and Eckstein, F: (1992) Proc. Natl. Acad. Sci. USA, 89, 918-921. (G5 and G8 cannnot have 2° F, 2' H, or 2' amino (reduction in k(cat) is about 150-fold for 2° F, 2' H, and about 10-fold for 2' amino). G12 can have 2' H and 2' amino, but cannot have 2° F.)

Yang, et al., (1990) Biochemistry 29:11156-11160.

Yang, J. -H., Usman, N., Chartrand, P. and Cedergren, R. (1992) Biochemistry, 31, 5005-5009. (DNA is OK at C3, T4, A6, T7, G12, A13, A14. Can't have DNA at G5, A15.1. If lots of sites are DNA, then rates drop dramatically if G8 is DNA or G15.2 is DNA.)

Zaug, A. J. et al, (1984), Science, 224:574-578

Zaug, A. J. & Cech, T. R., (1986A) Science, 231:473-474.

Zaug, A. J. et al., (1986B) Nature, 324:429-433. Zaug, A. J., and Cech, T. R. (1986) Biochemistry 25:4478.

Zhao, J. J. and Pick, L. (1993) Nature 365:448-451.

    __________________________________________________________________________     #             SEQUENCE LISTING                                                    - -  - - (1) GENERAL INFORMATION:                                              - -    (iii) NUMBER OF SEQUENCES: 47                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 13 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: RNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                - - GCGGGUCAUG AAG              - #                  - #                       - #      13                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 42 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                - - GACACUUCAU CUGAUGAGUC CUUUUGGACG AAACCCGCAG GT    - #                       - #  42                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                - - NCUGANGANG UUGAAAN             - #                  - #                       - #   17                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                - - NCUGANGANG UCGAAAN             - #                  - #                       - #   17                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 29 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                - - NNNNNNNCUG AUGAGTTTTG AAACNNNNN         - #                  - #                 29                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: RNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                - - UCCUGUCUUG CAUUG              - #                  - #                       - #    15                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 29 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                - - CAATGCAACU GAUGAGTTTT GAAACAGGA         - #                  - #                 29                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 29 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                - - CAATGCAACU GAUGAGTTTT GAAACAGGA         - #                  - #                 29                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 28 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                - - CAATGCAACU GACGACCTTG AAACAGGA         - #                  - #                  28                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 25 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                               - - CAATGCAACU GAUGANGAAA CAGGA          - #                  - #                    25                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 13 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                               - - GGAAGUCAGC CUA              - #                  - #                       - #      13                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 35 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                               - - GTCCTAGGCT CUGAUGAGTT TTGAAACTTC CTGGA       - #                  -      #       35                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                               - - GTCCTAGGCT CUGAUGATTT TGAAACTTCC TGGA       - #                  -       #        34                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                               - - UUCCAUGUCG GCAGAAT             - #                  - #                       - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                               - - ATTCTGCCCU GAUGAGTTTT GAAACATGGA A        - #                  - #               31                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 30 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                               - - ATTCTGCCCU GAUGATTTTG AAACATGGAA         - #                  - #                30                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                               - - ATTCTGCCCU GAUGAGGTTT GAAACATGGA A        - #                  - #               31                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                               - - CCAGGCAGUC AGAUCAUCUT            - #                  - #                       - # 20                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                               - - AAGATGATCT CUGAUGAGTT TTGAAACTGC CTGG       - #                  -       #        34                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 33 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                               - - AAGATGATCT CUGAUGATTT TGAAACTGCC TGG       - #                  -       #         33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                               - - NNNNNNNNNN NNNNNNN             - #                  - #                       - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                               - - ATTCTGCCGA CATGGAA             - #                  - #                       - #   17                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                               - - ATTCTGCCCU GAUGAGTTTT GAAACATGGA A        - #                  - #               31                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                               - - ATTCTGCCCU GAUGAGTTTT GAGACATGGA A        - #                  - #               31                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: RNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                               - - UCCUGUCUUG CAUUG              - #                  - #                       - #    15                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                               - - CAATGCAACU GAUGAGUCCU UUUGGACGAA ACAGGA      - #                  -      #       36                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 29 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                               - - CAATGCAACU GAUGAGTTTT GAAACAGGA         - #                  - #                 29                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 29 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                               - - CAATGCAACU GAUGAGTTTT GAGACAGGA         - #                  - #                 29                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                               - - CAATGCAAGA CAGGA              - #                  - #                       - #    15                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: DNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                               - - ACGAAAGACG CTAAG              - #                  - #                       - #    15                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 28 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                               - - CAATGCAACU GACGACCTTG AAACAGGA         - #                  - #                  28                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                               - - UCCUGUCUUG CAUUG              - #                  - #                       - #    15                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 29 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                               - - CAATGCAACU GAUGAGTTTC GAAACAGGA         - #                  - #                 29                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 30 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                               - - CAATGCAACU GAUGAGTTTT CGAAACAGGA         - #                  - #                30                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                               - - CAATGCAACU GAUGAGUCCU UUUGGACGAA ACAGGA      - #                  -      #       36                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                               - - CAATGCAACU GAUGAGTCCT TTTGGACGAA ACAGGA      - #                  -      #       36                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 30 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                               - - CAAUGCAACU GAUGAGUUUU CGAAACAGGA         - #                  - #                30                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                               - - CAAUGCAACU GAUGAGUCCU UUUGGACGAA ACAGGA      - #                  -      #       36                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 13 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: RNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                               - - GGAAGUCAGC CUA              - #                  - #                       - #      13                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: RNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                               - - UCCAGGAAGU CAGCCUAGGA            - #                  - #                       - # 20                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 35 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                               - - GTCCTAGGCT CUGAUGAGTT TCGAAACTTC CTGGA       - #                  -      #       35                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                               - - GTCCTAGGCT CUGAUGAGTT TTCGAAACTT CCTGGA      - #                  -      #       36                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 42 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                               - - GTCCTAGGCT CUGAUGAGTC CTTTTGGACG AAACTTCCTG GA    - #                       - #  42                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 13 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                               - - GCGAGUCCAC ACT              - #                  - #                       - #      13                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: RNA (genomic)                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                               - - AUUUGCGAGU CCACACUGGA G           - #                  - #                       - #21                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                               - - CTCCAGTGTG CUGAUGAGTT TTCGAAACTC GCAAAT      - #                  -      #       36                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 42 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: other nucleic acid                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                               - - CTCCAGTGTG CUGAUGAGTC CTTTTGGACG AAACTCGCAA AT    - #                       - #  42                                                                     __________________________________________________________________________ 

What is claimed is:
 1. A compound having the formula: ##STR30## wherein each X represents a nucleotide which may be the same or different and Q represents guanylate or deoxyguanylate and both of which may be substituted or modified in their sugar, base or phosphate;wherein (T/U)--(T/U)_(b) --Q represents T--(T)_(b) --Q or U--(U)_(b) --Q, with the proviso that b represents an integer which is 3 or 4; wherein each of (X)_(n-1) --A and _(n) (X) represents an oligonucleotide having a predetermined sequence which is capable of hybridizing with an RNA target sequence; wherein each of n and n' represents an integer which defines the number of nucleotides in the oligonucleotide such that (X)_(n-1) --A and (X)k_(n') are sufficient to hybridize to the target sequence; wherein each solid line represents a covalent bond between the nucleotides located on either side thereof; and wherein a represents an integer which defines a number of nucleotides with the proviso that a may be 0 or 1 and if 0, the A located 5' of (X)_(a) is bonded to the Q located 3' of (X)_(a).
 2. The compound of claim 1, wherein the oligonucleotide 3'--(X)_(n-1) --A is 3'--(X)_(n-2) --C--A--.
 3. The compound of claim 1, wherein (X)_(a) is absent.
 4. The compound of claim 1, wherein the integer b of (T/U)_(b) is equal to
 3. 5. The compound of claim 1, wherein the integer b of (T/U)_(b) is equal to
 4. 6. The compound of claim 1, wherein each X and Q is a deoxyribonucleotide.
 7. The compound of claim 1, wherein each X and Q is a ribonucleotide.
 8. The compound of claim 1, wherein (T/U)_(b) is a (T)_(b).
 9. The compound of claim 1, wherein (T/U)_(b) is (U)_(b).
 10. A compound having the formula: ##STR31## wherein each X represents a nucleotide which may be the same or different, P represents cytidylate or deoxycytidylate, Q represents guanylate or deoxyguanylate and each or which may be substituted or modified in their sugar, base or phosphate;wherein P--(T/U)_(b) --Q represents P--(T)_(b) --Q or P--(U)_(b) --Q, with the proviso that b represents an integer which is 3 or 4; wherein each of (X)_(n-1) --A and _(n) (X) represents an oligonucleotide having a predetermined sequence which is capable of hybridizing with an RNA target sequence; wherein each of n and n' represents an integer which defines the number of nucleotides in the oligonucleotide such that (X)_(n-1) --A and (X)_(n') are sufficient to hybridize to the target sequence; wherein * represents a base pair between the nucleotides located on either side thereof; wherein each solid line represents a covalent bond between the nucleotides located on either side thereof; and wherein a represents an integer which defines a number of nucleotides with the proviso that a may be 0 or 1 and if 0, the A located 5' of (X)_(a) is bonded to the Q located 3' of (X)_(a).
 11. The compound of claim 10, wherein the oligonucleotide 3'--(X)_(n-1) --A is 3'--(X)_(n-2) --CA--.
 12. The compound of claim 10, wherein (X)_(a) is absent.
 13. The compound of claim 10, wherein the integer b of (T/U)_(b) is equal to
 3. 14. The compound of claim 10, wherein the integer b of (T/U)_(b) is equal to
 4. 15. The compound of claim 10, wherein each X, P and Q is a deoxyribonucleotide.
 16. The compound of claim 10, wherein each X, P and Q is a ribonucleotide.
 17. The compound of claim 10, wherein (T/U)_(b) is a (T)_(b).
 18. The compound of claim 10, wherein (T/U)_(b) is a (U)_(b).
 19. A composition which comprises a compound of claim 1 in association with an acceptable carrier.
 20. A composition which comprises a compound of claim 10 in association with an acceptable carrier.
 21. A transfer vector containing a nucleotide sequence which on transcription gives rise to the compound of claim 1 or claim 10 wherein each X is a ribonucleotide, Q is a guanylate and P is a cytidylate.
 22. The transfer vector of claim 21, wherein the transfer vector is a bacterial plasmid, a bacteriophage DNA, a cosmid, or an eukaryotic viral DNA.
 23. The transfer vector of claim 21, wherein the oligonucleotide transfer vector is a plant DNA virus, a geminivirus or an infective phage particle.
 24. The transfer vector of claim 21, wherein the oligonucleotide transfer vector is packaged and contains the promoter sequences for RNA polymerase II or RNA polymerase III.
 25. A host cell transformed by the transfer vector of claim
 21. 26. The host cell of claim 25, wherein the host cell is a prokaryotic host cell or an eukaryotic host cell.
 27. The prokaryotic host cell of claim 26, wherein the prokaryotic host cell is an E. coli host cell.
 28. The eukaryotic host cell of claim 26, wherein the eukaryotic host cell is a monkey COS host cell, a Chinese hamster ovary host cell, a mammalian host cell or a plant host cell.
 29. A method of cleaving a target mRNA which comprises administering an effective amount of the compound of claim 1 or
 10. 30. A method of cleaving a target mRNA in a host cell which comprises administering to the host cell an effective amount of a compound of claim 1 or 10 or a transfer vector which on transcription expresses the compound.
 31. The compound of claim 1 or 10 which further comprises an antisense nucleic acid which is capable of hybridizing with an RNA target sequence.
 32. The compound of claim 1 or 10 which further comprises at least one additional non-naturally occurring oligonucleotide compound which comprises nucleotides whose sequence defines a conserved catalytic region and nucleotides whose sequence is capable of hybridizing with a predetermined target sequence.
 33. The compound of claim 32 wherein the additional non-naturally occurring oligonucleotide compound is a hammerhead ribozyme, a miniribozyme, a minizyme, a hairpin ribozyme, a hepatitis delta ribozyme, an RNAase P ribozyme or a combination thereof. 